IL-23p40 specific immunoglobulin derived proteins, compositions, epitopes, methods and uses

ABSTRACT

An anti-IL-23 specific human Ig derived protein, isolated nucleic acids that encode at least one anti-IL-23 Ig derived protein, vectors, host cells, transgenic animals or plants, and methods of making and using thereof are useful as therapeutic and diagnostic compositions, methods and devices. The anti-IL-23 Ig derived protein preferably binds to one or more of the Seg 1, Seg 2, and Seg 3 epitopes of the p40 subunit of IL-23.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.11/234,011, filed 23 Sep. 2005 now U.S. Pat. No. 7,252,971, which claimsthe benefit of priority to U.S. Provisional Application Ser. No.60/612,866, filed Sep. 24, 2004, and U.S. Provisional Application Ser.No. 60/616,832, filed Oct. 7, 2004. The entire contents of each of theforegoing applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to at least one IL-23p40 specific human Igderived protein or fragment thereof, encoding and complementary nucleicacids, host cells, IL-23p40 specific epitopes, and methods of making andusing thereof, including therapeutic formulations, administration anddevices.

BACKGROUND OF THE INVENTION

Interleukin-23 (IL-23) is the name given to a factor that is composed ofthe p40 subunit of IL-12 (IL-12beta, IL-12-p40) and another protein of19 kDa, designated p19. The p19 subunit is structurally related to IL-6,G-CSF, and the p35 subunit of IL-12. The p19 subunit by itself isbiologically inactive while the complex of p19 with p40 is active. Theactive complex is secreted by antigen presenting cells after cellactivation. Mouse memory T-cells (CD4 (+)CD45 Rb(low)) proliferate inresponse to IL-23 but not in response to IL-12. Human monocyte-derivedmacrophages produce IL-23 in response to virus infection (Sendai virus,but not Influenza A virus).

The IL-23 receptor complex consists of a receptor chain, termed IL23R,and the beta-1 subunit of the IL-12 receptor. IL-23 does not bind to thebeta-2 subunit of the IL-12 receptor. The human IL-23R gene is on humanchromosome 1 within 150 kb of the gene encoding IL-12Rbeta2. IL-23activates the same signaling molecules as IL-12: JAK2, Tyk2, and STAT-1,STAT-3, STAT-4, and STAT-5. STAT-4 activation is substantially weakerand different DNA-binding STAT complexes form in response to IL-23 ascompared to IL-12. IL-23R associates constitutively with JAK2 and in aligand-dependent manner with STAT-3.

Expression of p19 in transgenic mice leads to runting, systemicinflammation, infertility, and death before 3 months of age. The animalsshow high serum concentrations of the pro-inflammatory cytokinesTNF-alpha and IL-1. The number of circulating neutrophils is increased.Acute phase proteins are expressed constitutively. Animals expressing p19 specifically in the liver do not show these abnormalities. Expressionof p19 is most likely due to hematopoietic cells as bone marrowtransplantation of cells expressing p19 causes the same phenotype asthat observed in the transgenic animals.

Biologically active IL-12 exists as a heterodimer comprised of 2covalently linked subunits of 35 (p35) and 40 (p40) kD. IL-12 acts bybinding to both the IL-12beta 1 and beta 2 receptor proteins and therebyinduces signaling in a cell presenting both of these receptors. Severallines of evidence have demonstrated that IL-12 can induce robust Th1immune responses that are characterized by production of IFNγ and IL-2from CD4⁺ T cells. Inappropriate Th1 responses, and thus IL-12expression, are believed to correlate with many autoimmune diseases,such as multiple sclerosis, rheumatoid arthritis, inflammatory boweldisease, insulin-dependent diabetes mellitus, and uveitis. In animalmodels, IL-12 neutralization was shown to ameliorate autoimmune disease.However, these studies neutralized both IL-12 and IL-23 through theshared p40 subunit.

IL-12 is thought to be important in the development of Th1, or “type 1,”CD4+ T cell responses, whereas IL-23 is thought to be important for theactivation of memory CD4+ T cells. Several studies suggest that IL-12 isresponsible for generating effective immune responses to intracellularpathogens and tumor cells, while the function of IL-23 in these types ofimmune responses has yet to be fully described. Therefore, whileinhibition of both IL-12 and IL-23 should provide significant therapyfor immune-mediated disease, inhibition of IL-12 pathways could limitimmunity to pathogens or tumor cells and result in an unwanted riskprofile. In contrast, inhibition of only IL-23 could provide therapeuticbenefit while leaving IL-12 pathways intact. Thus, effective therapywould be achieved concomitant with a lowered risk profile.

Non-human, chimeric, polyclonal (e.g., anti-sera) and/or monoclonalantibodies (Mabs) and fragments (e.g., proteolytic digestion productsthereof) are potential therapeutic agents that are being developed insome cases to attempt to treat certain diseases. However, suchantibodies that comprise non-human portions elicit an immune responsewhen administered to humans. Such an immune response can result in animmune complex-mediated clearance of the antibodies from thecirculation, and make repeated administration unsuitable for therapy,thereby reducing the therapeutic benefit to the patient and limiting thereadministration of the Ig derived protein. For example, repeatedadministration of antibodies comprising non-human portions can lead toserum sickness and/or anaphalaxis. In order to avoid these and othersuch problems, a number of approaches have been taken to reduce theimmunogenicity of such antibodies and portions thereof, includingchimerization and “humanization,” as well known in the art. Theseapproaches have produced antibodies having reduced immunogenicity, butwith other less desirable properties.

Accordingly, there is a need to provide anti-IL-23 antibodies orspecified portions or variants, nucleic acids, host cells, compositions,and methods of making and using thereof, that overcome one more of theseproblems.

SUMMARY OF THE INVENTION

The present invention provides immunoglobulin (Ig) derived proteins thatare specific for the p40 subunit of IL-23, (“anti-IL-23 Ig derivedprotein” or “IL-23 Ig derived protein”). Preferably, the IL-23 Igproteins do not bind to the p40 subunit of IL-12, and/or inhibit bindingof IL-12 to one or more of its receptors. Such Ig derived proteins,including antibodies, antagonists or receptor fusion proteins block thebinding of IL-23 to at least one of its receptors by binding to the p40subunit of IL-23. For example, they bind to human IL-23p40 and blockbinding of human IL-23 to at least one of its receptors. The presentinvention further provides compositions, formulations, methods, devicesand uses of such anti-IL-23 Ig derived proteins, including fortherapeutic and diagnostic uses.

In one embodiment, the present invention provides the predicted epitoperegions for the human and murine IL-23p40 subunits. These epitopes arepredicted to be available for binding on the IL-23p40 subunit (of theIL-23 dual subunit protein) and unavailable on the IL-12p40 subunit(alone or of the IL-12 dual subunit protein).

In a further embodiment, the present invention provides Ig derivedproteins that selectively inhibit IL-23 related activities and, furtherdo not inhibit IL-12 specific activities that are mediated by thebinding of IL-12 to one or more of its receptors.

In another embodiment, the present invention provides Ig derivedproteins that inhibit IL-23 activity in antigen presenting cells (APCs),such as but not limited to, macrophages, microglia, mesangialphagocytes, synovial A cells, stem cell precursors, Langerhans cells,Kuppfer cells, dendritic cells, B cells, and the like. Such APCs can bepresent in different tissues, e.g., but not limited to, skin, epidermis,liver, spleen, brain, spinal cord, thymus, bone marrow, joint synovialfluid, kidneys, blood, and the like. Such APCs can also be limited tooutside or inside the blood brain barrier.

In a further embodiment, the present invention provides Ig derivedproteins that are suitable for treating at least one IL-23 relatedcondition by blocking IL-23 binding to one or more of its receptors,and, optionally, where the Ig derived proteins do not block IL-12binding to one or more of its receptors such that no independent IL-12function is impaired.

The present invention thus provides isolated human, primate, rodent,mammalian, chimeric, humanized and/or CDR-grafted anti-IL-23 Ig derivedproteins (Ig derived proteins), including antibodies, immunoglobulins,receptor fusion proteins, cleavage products and other specified portionsand variants thereof, as well as anti-IL-23 Ig derived proteincompositions, encoding or complementary nucleic acids, vectors, hostcells, compositions, formulations, devices, transgenic animals,transgenic plants, and methods of making and using thereof, as describedand enabled herein, in combination with what is known in the art. Suchanti-IL-23 Ig derived proteins act as antagonists to IL-23 proteinsand/or IL-23 receptors and thus are useful for treating IL-23pathologies. IL-23 proteins include, but are not limited to, IL-23,particularly, the p40 subunit of IL-23, as well as the p19 subunit ofIL-23.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding specific IL-23 Ig derived proteins or specified portions orvariants thereof, comprising at least one specified sequence, domain,portion or variant thereof. The present invention further providesrecombinant vectors comprising said isolated IL-23 Ig derived proteinnucleic acid molecules, host cells containing such nucleic acids and/orrecombinant vectors, as well as methods of making and/or using such Igderived protein nucleic acids, vectors and/or host cells.

At least one Ig derived protein or specified portion or variant of theinvention binds at least one specified epitope specific to at least oneIL-23 protein, receptor, subunit, fragment, portion or any combinationthereof. The at least one epitope can comprise at least one Ig derivedprotein binding region that comprises at least one portion of the matureor the extracellular region of said protein and/or receptor.Non-limiting examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13or 14 amino acids of at least one of, 1-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220,220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300,300-306, 1-7, 14-21, 29-52, 56-73, 83-93, 96-105, 156-175, 194-204,208-246, 254-273, 279-281, or 289-300 of SEQ ID NO: 1 as the human p40subunit (306 amino acids). Preferred examples include 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 amino acids of the mature peptide or extracellular domainof human IL-23p40 (SEQ ID NO: 1). More specifically, the epitope regionsare predicted to comprise all or a portion of Seg 1 (amino acids 97-101of SEQ ID NO: 1), and/or Seg 2 (amino acids 138-145 of SEQ ID NO:1),and/or Seg 3 (amino acids 250-269 of SEQ ID NO:1) of SEQ ID NO:1. Themurine counterparts (on the murine p40 subunit) for these epitoperegions are Murineseg 1 (amino acids 97-98 of the murine sequence shownin Table 5 below without the signal sequence or amino acids 119-120 ofthe sequence containing the 22-amino acid signal sequence), Murineseg 2(amino acids 135-142 of the murine sequence shown in Table 5 belowwithout the signal sequence or amino acids 157-164 of the sequencecontaining the 22-amino acid signal sequence), Murineseg 3 (amino acids247-274 of the murine sequence shown in Table 5 below without the signalsequence or amino acids 269-296 of the sequence containing the 22-aminoacid signal sequence). The at least one Ig derived protein or specifiedportion or variant amino acid sequence can further optionally compriseat least one specified substitution, insertion or deletion.

The present invention also provides at least one composition comprising(a) an isolated IL-23 Ig derived protein or specified portion or variantencoding nucleic acid and/or Ig derived protein as described herein; and(b) a suitable carrier or diluent. The carrier or diluent can optionallybe pharmaceutically acceptable, according to known methods. Thecomposition can optionally further comprise at least one other compound,protein or composition.

The present invention also provides at least one method for expressingat least one IL-23 Ig derived protein or specified portion or variant ina host cell, comprising culturing a host cell as described herein and/oras known in the art under conditions wherein at least one IL-23 Igderived protein or specified portion or variant is expressed indetectable and/or recoverable amounts.

The present invention further provides at least one IL-23 Ig derivedprotein, specified portion or variant in a method or composition, whenadministered in a therapeutically effective amount, for modulation, fortreating or reducing the symptoms of immune, neurological, and relateddisorders, such as, but not limited to, multiple sclerosis, rheumatoidarthritis, juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis,gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatorybowel disease, ulcerative colitis, systemic lupus erythematosis,antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis,idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever,perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria,systemic anaphalaxis, dermatitis, pernicious anemia, hemolyticdisesease, thrombocytopenia, graft rejection of any organ or tissue,kidney transplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to, asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, acute or chronic bacterialinfection, acute and chronic parasitic or infectious processes,including bacterial, viral and fungal infections, HIV infection/HIVneuropathy, meningitis, hepatitis (e.g., A, B or C, or the like), septicarthritis, peritonitis, pneumonia, epiglottitis, e. Coli 0157:h7,hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shocksyndrome, streptococcal myositis, gas gangrene, mycobacteriumtuberculosis, mycobacterium avium intracellulare, pneumocystis cariniipneumonia, pelvic inflammatory disease, orchitis/epidydimitis,legionella, lyme disease, influenza a, epstein-barr virus,vital-associated hemaphagocytic syndrome, vital encephalitis/asepticmeningitis, neurodegenerative diseases, multiple sclerosis, migraineheadache, AIDS dementia complex, demyelinating diseases, such asmultiple sclerosis and acute transverse myelitis; extrapyramidal andcerebellar disorders, such as lesions of the corticospinal system;disorders of the basal ganglia; hyperkinetic movement disorders, such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multi.system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit, such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,neurotraumatic injury (e.g., but not limited to, spinal cord injury,brain injury, concussion, and repetitive concussion), pain, inflammatorypain, autism, depression, stroke, cognitive disorders, epilepsy, and thelike, as needed in many different conditions, such as but not limitedto, prior to, subsequent to, or during a related disease or treatmentcondition, as known in the art.

The present invention further provides at least one IL-23 Ig derivedprotein, specified portion or variant in a method or composition, whenadministered in a therapeutically effective amount, for modulation, fortreating or reducing the symptoms of at least one IL-23 disease in acell, tissue, organ, animal or patient and/or, as needed in manydifferent conditions, such as but not limited to, prior to, subsequentto, or during a related disease or treatment condition, as known in theart and/or as described herein.

The present invention also provides at least one composition, deviceand/or method of delivery of a therapeutically or prophylacticallyeffective amount of at least one IL-23 Ig derived protein or specifiedportion or variant, according to the present invention.

The present invention also provides at least one isolated IL-23 Igderived protein, comprising at least one immunoglobulin complementaritydetermining region (CDR) or at least one ligand binding region (LBR)that specifically binds at least one IL-23 protein or receptor, whereinsaid IL-23 Ig derived protein specifically binds at least one epitopenot available on the IL-12p40 subunit comprising at least 1-3 aminoacids of the entire amino acid sequence (amino acids 1-306) of theIL-23p40 subunit (SEQ ID NO:1), such as but not limited to, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids of at least one of,1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100,100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180,180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260,260-270, 280-290, 290-300, 300-306, 1-7, 14-21, 29-52, 56-73, 83-93,96-105, 156-175, 194-204, 208-246, 254-273, 279-281, or 289-300 of SEQID NO:1. Preferred examples include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids of the mature peptide or extracellular domain of humanIL-23p40 (SEQ ID NO:1), comprised of SEQ ID NO:1, more specifically, theepitope regions are predicted to comprise Seg 1 (amino acids 97-101 ofSEQ ID NO:1), and/or Seg 2 (amino acids 138-145 of SEQ ID NO:1), and/orSeg 3 (amino acids 250-269 of SEQ ID NO:1) of SEQ ID NO:1.

The invention also provides at least one isolated IL-23 human Ig derivedprotein encoding nucleic acid, comprising a nucleic acid that hybridizesunder stringent conditions, or has at least 95% identity, to a nucleicacid encoding an IL-23 Ig derived protein. The invention furtherprovides an isolated IL-23 human Ig derived protein, comprising anisolated human Ig derived protein encoded by such a nucleic acid. Theinvention further provides an IL-23 human Ig derived protein encodingnucleic acid composition, comprising such an isolated nucleic acid and acarrier or diluent. The invention further provides an Ig derived proteinvector, comprising such a nucleic acid, wherein the vector optionallyfurther comprises at least one promoter selected from the groupconsisting of a late or early SV40 promoter, a CMV promoter, an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, a humanimmunoglobulin promoter, and an EF-1 alpha promoter. Such a vector canoptionally further comprise at least one selection gene or portionthereof selected from at least one of methotrexate (MTX), dihydrofolatereductase (DHFR), green fluorescent protein (GFP), neomycin (G418), orglutamine synthetase (GS). The invention further comprises a mammalianhost cell comprising such an isolated nucleic acid, optionally, whereinsaid host cell is at least one selected from COS-1, COS-7, HEK293,BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphomacells, or any derivative, immortalized or transformed cell thereof.

The invention also provides at least one method for producing at leastone IL-23 human Ig derived protein, comprising translating such anucleic acid or an endogenous nucleic acid that hybridizes thereto understringent conditions, under conditions in vitro, in vivo or in situ,such that the IL-23 human Ig derived protein is expressed in detectableor recoverable amounts.

The invention also provides at least one IL-23 human Ig derived proteincomposition, comprising at least one isolated IL-23 human Ig derivedprotein and a carrier or diluent, optionally, further wherein saidcarrier or diluent is pharmaceutically acceptable, and/or furthercomprising at least one compound or protein selected from at least oneof a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an IL-23 agent, a mineral, a nutritional, a thyroid agent, a vitamin, acalcium related hormone, an antidiarrheal, an antitussive, anantiemetic, an antiulcer, a laxative, an anticoagulant, anerythropieitin, a filgrastim, a sargramostim, an immunization, animmunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, an antimanic agent,an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha, a cytokine, and a cytokineantagonist.

The present invention also provides at least one method for treating anIL-23 condition in a cell, tissue, organ or animal, comprisingcontacting or administering a immune related- or infectiousrelated-condition modulating effective amount of at least one IL-23human Ig derived protein with, or to, said cell, tissue, organ oranimal, optionally, wherein said animal is a primate, optionally amonkey or a human. The method can further optionally include whereinsaid effective amount is about 0.001-100 mg/kilogram of said cells,tissue, organ or animal. Such a method can further include wherein saidcontacting or said administrating is by at least one mode selected fromintravenous, intramuscular, bolus, intraperitoneal, subcutaneous,respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual,intranasal, subdermal, and transdermal. Such a method can furthercomprise administering, prior, concurrently or after said contacting oradministering, at least one composition comprising a therapeuticallyeffective amount of at least one compound or protein selected from atleast one of a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an IL-23 agent, a mineral, a nutritional, a thyroid agent, a vitamin, acalcium related hormone, an antidiarrheal, an antitussive, anantiemetic, an antiulcer, a laxative, an anticoagulant, anerythropoietin, a filgrastim, a sargramostim, an immunization, animmunoglobulin, an immunosuppressive, a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, an antimanic agent,an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha, a cytokine, and a cytokineantagonist.

The present invention also provides at least one medical device,comprising at least one IL-23 human Ig derived protein, wherein saiddevice is suitable for contacting or administering said at least oneIL-23 human Ig derived protein by at least one mode selected fromintravenous, intramuscular, bolus, intraperitoneal, subcutaneous,respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual,intranasal, sub dermal, and trans dermal.

The present invention also provides at least one human immunoglobulinlight chain IL-23 protein, comprising at least one portion of a variableregion comprising at least one human Ig derived protein fragment of theinvention.

The present invention also provides at least one human immunoglobulinheavy chain or portion thereof, comprising at least one portion of avariable region comprising at least one IL-23 human Ig derived proteinfragment.

The invention also includes at least one human Ig derived protein,wherein said human Ig derived protein binds the same epitope orantigenic region as an IL-23 human Ig derived protein.

The invention also includes at least one formulation comprising at leastone IL-23 human Ig derived protein, and at least one selected fromsterile water, sterile buffered water, or at least one preservativeselected from the group consisting of phenol, m-cresol, p-cresol,o-cresol, chlorocresol, benzyl alcohol, alkylparaben, benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent, optionally, wherein theconcentration of IL-23 human Ig derived protein is about 0.1 mg/ml toabout 100 mg/ml, further comprising at least one isotonicity agent or atleast one physiologically acceptable buffer.

The invention also includes at least one formulation comprising at leastone IL-23 human Ig derived protein in lyophilized form in a firstcontainer, and an optional second container comprising at least one ofsterile water, sterile buffered water, or at least one preservativeselected from the group consisting of phenol, m-cresol, p-cresol,o-cresol, chlorocresol, benzyl alcohol, alkylparaben, benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent, optionally further whereinthe concentration of IL-23 human Ig derived protein is reconstituted toa concentration of about 0.1 mg/ml to about 500 mg/ml, furthercomprising an isotonicity agent, or further comprising a physiologicallyacceptable buffer.

The invention further provides at least one method of treating at leastone IL-23 mediated condition, comprising administering to a patient inneed thereof a formulation of the invention.

The invention also provides at least one article of manufacture forhuman pharmaceutical use, comprising packaging material and a containercomprising a solution or a lyophilized form of at least one IL-23 humanIg derived protein of the invention, optionally, further wherein saidcontainer is a glass or plastic container having a stopper for multi-useadministration, optionally, further wherein said container is a blisterpack, capable of being punctured and used in intravenous, intramuscular,bolus, intraperitoneal, subcutaneous, respiratory, inhalation, nasal,vaginal, rectal, buccal, sublingual, intranasal, subdermal, ortransdermal administration; said container is a component of anintravenous, intramuscular, bolus, intraperitoneal, subcutaneous,respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual,intranasal, subdermal, or transdermal delivery device or system; saidcontainer is a component of an injector or pen-injector device or systemfor intravenous, intramuscular, bolus, intraperitoneal, subcutaneous,respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual,intranasal, sub dermal, or trans dermal delivery.

The invention further provides at least one method for preparing aformulation of at least one IL-23 human Ig derived protein of theinvention, comprising admixing at least one IL-23 human Ig derivedprotein in at least one buffer containing saline or a salt.

The invention also provides at least one method for producing at leastone IL-23 human Ig derived protein of the invention, comprisingproviding a host cell, transgenic animal, transgenic plant or plant cellcapable of expressing in recoverable amounts said human Ig derivedprotein, optionally, further wherein said host cell is a mammalian cell,a plant cell or a yeast cell; said transgenic animal is a mammal; saidtransgenic mammal is selected from a goat, a cow, a sheep, a horse, anda non-human primate.

The invention further provides at least one transgenic animal or plantexpressing at least one human Ig derived protein of the invention.

The invention further provides at least one IL-23 human Ig derivedprotein produced by a method of the invention.

The invention further provides at least one method for treating at leastone IL-23 mediated disorder, comprising at least one of (a)administering an effective amount of a composition or pharmaceuticalcomposition comprising at least one IL-23 human Ig derived protein to acell, tissue, organ, animal or patient in need of such modulation,treatment or therapy; and (b) further administering, before,concurrently, and/or after said administering in (a) above, at least oneselected from an immune related therapeutic, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, a neurologicalagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an antidiarrheal, an antitussive, an antiemetic, anantiulcer, a laxative, an anticoagulant, an erythropoietin, afilgrastim, a sargramostim, an immunizing agent, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, anestrogen receptor modulator, a mydriatic, a cycloplegic, an alkylatingagent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, adonepezil, a tacrine, anasthma medication, a beta agonist, an inhaled steroid, a leukotrieneinhibitor, a methylxanthine, a cromolyn, an epinephrine or analog, adomase alpha, a cytokine, and a cytokine antagonist.

The present invention further comprises any invention described hereinand is not limited to any particular description, embodiment or exampleprovided herein.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A is a graph showing the specificity of the anti-IL-23 antibodyfor IL-23.

FIG. 1B is a graph showing the specificity of the anti-IL-23 antibodyfor the IL-23p40 subunit.

FIG. 1C is a graph showing the biological effect of antibodies on IL-23induced IL-17 production.

FIG. 1D is a graph showing the biological effect of antibodies on IL-12induced IFNγ production.

FIG. 1E is a graph showing the clinical suppression of EAE by theantibodies.

FIG. 2A is an image showing the H&E stained thoracic spinal cord frommice treated with control rat IgG (40 times magnification).

FIG. 2B is an image showing the H&E stained thoracic spinal cord frommice treated with anti-IL-23 antibody (40 times magnification).

FIG. 2C is an image showing the Luxol Blue stained thoracic spinal cordfrom mice treated with control rat IgG (200 times magnification).

FIG. 2D is an image showing the Luxol Blue stained thoracic spinal cordfrom mice treated with anti-IL-23 antibody (200 times magnification).

FIG. 2E is an image showing the GFAP stained thoracic spinal cord frommice treated with control rat IgG (200 times magnification).

FIG. 2F is an image showing the GFAP stained thoracic spinal cord frommice treated with anti-IL-23 antibody (200 times magnification).

FIG. 3 shows the understood mechanisms for specific antibodyneutralization of IL-12 and/or IL-23.

FIG. 4 illustrates an example of an IL-12RP 1 binding p40 epitope onlyavailable on IL-23.

FIG. 5 shows the crystal structure of IL-12p40/p35.

FIG. 6A shows the effect of anti-IL-23 and anti-p40 antibodies on EAEprogression.

FIG. 6B shows the effect of anti-IL-12 and anti-p40 antibodies on L.Major infection.

FIG. 6C shows the effect of anti-IL-12, anti-IL-23, and anti-p40antibodies on L. Major infection.

FIG. 7 shows the results of an ELISA evaluation of CNTO 1836 binding towild-type human and murine IL-23 and mutated forms of human and murineIL-23.

FIG. 8 shows the results of an ELISA evaluation of CNTO 1275 binding towild-type human and murine IL-23 and mutated forms of human and murineIL-23.

DESCRIPTION OF THE INVENTION

The present invention provides immunoglobulin (Ig) derived proteins thatare specific for the p40 subunit of IL-23 and which preferably do notbind to the p40 subunit of IL-12 or the IL-12 beta 2 receptor. Such Igderived proteins include antibody and receptor fusion proteins thatblock the binding of IL-23 to at least one of its receptors (e.g., butnot limited to, IL-23 receptor and/or IL-12 beta 1 receptor) by bindingto the p19 or p40 subunits of IL-23 and/or the IL-23 receptors.Preferably, such anti-IL-23 Ig derived proteins do not bind and/orinhibit binding of IL-12 to one or more of its receptors, e.g., but notlimited to IL-12 beta 1 receptor and/or IL-12 beta 2 receptor. Thepresent invention further provides compositions, formulations, methods,devices and uses of such anti-IL-23 Ig derived proteins, including fortherapeutic and diagnostic uses.

The present invention also provides Ig derived proteins that selectivelyinhibit IL-23 related activities and, further, do not inhibit IL-12specific activities that are mediated by the binding of IL-12 to one ormore of its receptors (e.g., but not limited to, IL-12 beta 1 receptor,or IL-12 beta 2 receptor).

The present invention further provides Ig derived proteins that aresuitable for treating at least one IL-23 related condition by blockingIL-23 binding to one or more of its receptors, and where the Ig derivedproteins do not block IL-12 binding to one or more of its receptors.

The present invention also provides Ig derived proteins that inhibitIL-23 activity in antigen presenting cells (APCs), such as but notlimited to, macrophages, microglia, mesangial phagocytes, synovial Acells, stem cell precursors, Langerhans cells, Kuppfer cells, dendriticcells, B cells, and the like. Such APC's can be present in differenttissues, e.g., but not limited to, skin, epidermis, liver, spleen,brain, spinal cord, thymus, bone marrow, joint synovial fluid, kidneys,blood, and the like. Such APC's can also be limited to outside or insidethe blood brain barrier.

The present invention provides isolated, recombinant and/or syntheticIL-23 Ig derived proteins or specified portions or variants, as well ascompositions and encoding nucleic acid molecules comprising at least onepolynucleotide encoding at least one IL-23 Ig derived protein. Such Igderived proteins or specified portions or variants of the presentinvention comprise specific full length Ig derived protein sequences,domains, fragments and specified variants thereof, and methods of makingand using said nucleic acids and Ig derived proteins or specifiedportions or variants, including therapeutic compositions, methods anddevices.

As used herein, an “anti-IL-23 Ig derived protein,” “anti-IL-23 Igderived protein portion,” “anti-IL-23 Ig derived protein fragment,”“anti-IL-23 Ig derived protein variant,” “IL-23 Ig derived protein,”“IL-23 Ig derived protein portion,” or “IL-23 Ig derived proteinfragment” and/or “IL-23 Ig derived protein variant” and the likedecreases, blocks, inhibits, abrogates or interferes with IL-23 proteinactivity, binding or IL-23 protein receptor activity or binding invitro, in situ and/or, preferably, in vivo. As used herein, “IL-12p40”or “IL-23p40” refers to the p40 subunit of IL-23, as well as activeportions, fragments, isoforms, splice variants, and the like, as knownin the art

For example, a suitable IL-23 Ig derived protein, specified portion orvariant of the present invention can bind at least one IL-23 protein orreceptor and includes anti-IL-23 Ig derived proteins, antigen-bindingfragments thereof, and specified portions, variants or domains thereofthat bind specifically to IL-23 and/or one or more of its receptors. Asuitable IL-23 Ig derived protein, specified portion, or variant canalso decrease, block, abrogate, interfere, prevent and/or inhibit IL-23protein RNA, DNA or protein synthesis, IL-23 protein release, IL-23protein or receptor signaling, membrane IL-23 protein cleavage, IL-23related activity, IL-23 protein production and/or synthesis, e.g., asdescribed herein or as known in the art.

Anti-IL-23 Ig derived proteins (also termed anti-IL-23 Ig derivedproteins) useful in the methods and compositions of the presentinvention are characterized by high affinity binding to IL-23 proteinsand/or receptors, and, optionally and preferably, having low toxicity.In particular, an Ig derived protein, specified fragment or variant ofthe invention, where the individual components, such as the variableregion, constant region and framework, individually and/or collectively,optionally and preferably possess low immunogenicity, is useful in thepresent invention. The Ig derived proteins that can be used in theinvention are optionally characterized by their ability to treatpatients for extended periods with good to excellent alleviation ofsymptoms and low toxicity. Low immunogenicity and/or high affinity, aswell as other suitable properties, may contribute to the therapeuticresults achieved. “Low immunogenicity” is defined herein as raisingsignificant HAHA, HACA or HAMA responses in less than about 75%, orpreferably less than about 50% of the patients treated and/or raisinglow titres in the patient treated (less than about 300, preferably, lessthan about 100 measured with a double antigen enzyme immunoassay)(Elliott et al., Lancet 344:1125-1127 (1994)), each of the abovereferences entirely incorporated herein by reference.

Utility

The isolated nucleic acids of the present invention can be used forproduction of at least one IL-23 Ig derived protein, fragment orspecified variant thereof, which can be used to effect in an cell,tissue, organ or animal (including mammals and humans), to modulate,treat, alleviate, help prevent the incidence of, or reduce the symptomsof, at least one IL-23 condition.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-IL-23 Ig derived protein or specified portion or variant to a cell,tissue, organ, animal or patient in need of such modulation, treatment,alleviation, prevention, or reduction in symptoms, effects ormechanisms. The effective amount can comprise an amount of about 0.001to 500 mg/kg per single or multiple administration, or to achieve aserum concentration of 0.01-5000 μg/ml serum concentration per single ormultiple administration, or any effective range or value therein, asdone and determined using known methods, as described herein or known inthe relevant arts.

Citations

All publications or patents cited herein are entirely incorporatedherein by reference as they show the state of the art at the time of thepresent invention and/or to provide description and enablement of thepresent invention. Publications refer to any scientific or patentpublications, or any other information available in any media format,including all recorded, electronic or printed formats. In addition tothose specified herein, the following references are entirelyincorporated herein by reference: Ausubel, et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.(1987-2003); Sambrook, et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Igderived proteins, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989);Colligan, et al., eds., Current Protocols in Immunology, John Wiley &Sons, Inc., NY (1994-2003); Colligan et al., Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2003).

Ig Derived Proteins of the Present Invention

The term “Ig derived protein” is intended to encompass Ig derivedproteins, digestion fragments, specified portions and variants thereof,including Ig derived protein mimetics or comprising portions of Igderived proteins that mimic the structure and/or function of an antibodyor specified fragment or portion thereof, including single chain Igderived proteins and fragments thereof, and is also intended toencompass proteins that contain mimetics to therapeutic proteins,antibodies, and digestion fragments, specified portions and variantsthereof, wherein the protein comprises at least one functional IL-23protein or receptor ligand binding region (LBR) that optionally replacesat least one complementarity determining region (CDR) of the antibodyfrom which the Ig-derived protein, portion or variant is derived. SuchIL-23 IgG derived proteins, specified portions or variants include thosethat mimic the structure and/or function of at least one IL-23 proteinantagonist, such as an IL-23 protein antibody, receptor, ligand protein,fragment or analog. Functional fragments include antigen-bindingfragments that bind to human IL-23 proteins, receptors, or fragmentsthereof. For example, Ig derived protein fragments capable of binding tohuman IL-23 proteins, receptors, or fragments thereof, including, butnot limited to, Fab (e.g., by papain digestion), Fab′ (e.g., by pepsindigestion and partial reduction) and F(ab′)₂ (e.g., by pepsindigestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin orplasmin digestion), Fd (e.g., by pepsin digestion, partial reduction andreaggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are encompassed by the invention (see, e.g., Colligan,Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Ig derived proteins can also be produced in a variety of truncated formsusing Ig derived protein genes in which one or more stop codons havebeen introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain portion can be designed toinclude DNA sequences encoding the CH₁, domain and/or hinge region ofthe heavy chain. The various portions of Ig derived proteins can bejoined together chemically by conventional techniques, or can beprepared as a contiguous protein using genetic engineering techniques.For example, a nucleic acid encoding the variable and constant regionsof a human Ig derived protein chain can be expressed to produce acontiguous protein. See, e.g., Colligan, Immunology, supra, sections 2.8and 2.10, for fragmentation and Ladner et al., U.S. Pat. No. 4,946,778and Bird, R. E. et al., Science, 242: 423-426 (1988), regarding singlechain Ig derived proteins, each of which publications are entirelyincorporated herein by reference.

As used herein, the term “human Ig derived protein” refers to an Igderived protein in which substantially every part of the protein (e.g.,CDR, LBR, framework, C_(L), C_(H) domains (e.g., C_(H)1, C_(H)2,C_(H)3), hinge, (V_(L), V_(H))) is substantially non-immunogenic, withonly minor sequence changes or variations. Such changes or variationsoptionally and preferably retain or reduce the immunogenicity in humansrelative to non-modified human Ig derived proteins. Thus, a human Igderived protein is distinct from a chimeric or humanized Ig. It ispointed out that a human Ig derived protein can be produced by anon-human animal or prokaryotic or eukaryotic cell that is capable ofexpressing functionally rearranged human immunoglobulin (e.g., heavychain and/or light chain) genes. Further, when a human Ig derivedprotein is a single chain Ig derived protein, it can comprise a linkerpeptide that is not found in native human Ig derived proteins. Forexample, an Fv can comprise a linker peptide, such as two to about eightglycine or other amino acid residues, which connects the variable regionof the heavy chain and the variable region of the light chain. Suchlinker peptides are considered to be of human origin. IL-23 Ig derivedproteins that comprise at least one IL-23 protein ligand or receptorthereof can be designed against an appropriate ligand, such as anisolated and/or IL-23 protein, receptor, or a portion thereof (includingsynthetic molecules, such as synthetic peptides). Preparation of suchIL-23 Ig derived proteins are performed using known techniques toidentify and characterize ligand binding regions or sequences of atleast one IL-23 protein, receptor, or portion thereof.

Human Ig derived proteins that are specific for p40 subunit and/or IL-23receptors can be raised against an appropriate immunogenic antigen, suchas isolated IL-23 protein, receptor, or a portion thereof (includingsynthetic molecules, such as synthetic peptides). Preparation ofimmunogenic antigens, and monoclonal Ig derived protein production canbe performed using any suitable technique. A variety of methods havebeen described (see e.g., Kohler et al., Nature, 256: 495-497 (1975) andEur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552(1977); Koprowski et al, U.S. Pat. No. 4,172,124; Harlow, E. and D.Lane, 1988, Ig derived proteins: A Laboratory Manual, (Cold SpringHarbor Laboratory: Cold Spring Harbor, N.Y.); Current Protocols InMolecular Biology, Vol. 2 (e.g., Supplement 27, Summer '94), Ausubel, F.M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11,(1991-2003)), each of which is entirely incorporated herein byreference. Generally, a hybridoma is produced by fusing a suitableimmortal cell line (e.g., a myeloma cell line such as, but not limitedto, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1,JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO2A, or the like, or heteromyelomas, fusion products thereof, or any cellor fusion cell derived therefrom, or any other suitable cell line asknown in the art, see, e.g., www.atcc.org, www.lifetech.com., and thelike, each of which is entirely incorporated herein by reference) withIg derived protein producing cells, such as, but not limited to,isolated or cloned spleen cells, or any other cells expressing heavy orlight chain constant, variable, framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2, eachentirely incorporated herein by reference.

Ig derived protein producing cells can be obtained from the peripheralblood or, preferably, the spleen or lymph nodes, of humans or othersuitable animals that have been immunized with the antigen of interest.Any other suitable host cell can also be used for expressingheterologous or endogenous nucleic acid encoding an Ig derived protein,specified fragment or variant thereof, of the present invention. Thefused cells (hybridomas) or recombinant cells can be isolated usingselective culture conditions or other suitable known methods, and clonedby limiting dilution or cell sorting, or other known methods. Cellswhich produce Ig derived proteins with the desired specificity can beselected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, Del.; Biovation, Aberdeen, Scotland, UK;BioInvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260(May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); WO96/13583, WO97/08320(MorphoSys); WO95/16027 (BioInvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirelyincorporated herein by reference) or that rely upon immunization oftransgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol.41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118(1996); Eren et al., Immunol. 93:154-161 (1998), each entirelyincorporated by reference as well as related patents and applications)that are capable of producing a repertoire of human antibodies, as knownin the art and/or as described herein. Such techniques, include, but arenot limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,95:14130-14135 (November 1998)); single cell antibody producingtechnologies (e.g., selected lymphocyte antibody method (“SLAM”) (U.S.Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gelmicrodroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337(1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. 1 mm. Meth.182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995));B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134(1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization inHybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V.,Amsterdam, Netherlands (1988)), each of which is entirely incorporatedherein by reference.

Methods for humanizing non-human Ig derived proteins can also be usedand are well known in the art. Generally, a humanized antibody has oneor more amino acid residues introduced into it from a source that isnon-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an “import” variabledomain. Humanization can be essentially performed following the methodof Winter and co-workers (Jones et al., Nature 321:522 (1986); Riechmannet al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534(1988), each of which is entirely incorporated herein by reference), bysubstituting rodent CDRs for CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” Ig derivedproteins are chimeric Ig derived proteins (Cabilly et al., supra),wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized Ig derived proteins are typically human Ig derivedproteins in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent Ig derivedproteins.

The choice of human variable domains, both light and heavy, to be usedin making the humanized Ig derived proteins can be used to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), each of which is entirely incorporated herein by reference).Another method uses a particular framework derived from the consensussequence of all human Ig derived proteins of a particular subgroup oflight or heavy chains. The same framework can be used for severaldifferent humanized Ig derived proteins (Carter et al., Proc. Natl.Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623(1993), each of which is entirely incorporated herein by reference).

Ig derived proteins can also optionally be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanized Igderived proteins are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Human monoclonal Ig derived proteins can be made by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal Ig derived proteins have been described,for example, by Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.147:86 (1991), each of which is entirely incorporated herein byreference.

Alternatively, phage display technology, in addition to that presentedabove, can be used to produce human Ig derived proteins and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to one non-limitingexample of this technique, antibody V domain genes are cloned in-frameinto either a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats; for their reviewsee, e.g., Johnson et al., Current Opinion in Structural Biology 3:564(1993), each of which is entirely incorporated herein by reference.

Several sources of V-gene segments can be used for phage display.Clackson et al., Nature 352:624 (1991) isolated a diverse array ofanti-oxazolone Ig derived proteins from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand Ig derived proteins to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., J. Mol. Biol. 222:581 (1991), or Griffith etal., EMBO J. 12:725 (1993), each of which is entirely incorporatedherein by reference.

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (Marks et al.,Bio/Technol. 10:779 (1992)). In this method, the affinity of “primary”human Ig derived proteins obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of Ig derived proteins and antibody fragments with affinitiesin the nM range.

A strategy for making very large phage antibody repertoires has beendescribed by Waterhouse et al., Nucl. Acids Res. 21:2265 (1993). Geneshuffling can also be used to derive human Ig derived proteins fromrodent Ig derived proteins, where the human antibody has similaraffinities and specificities to the starting rodent antibody. Accordingto this method, which is also referred to as “epitope imprinting,” theheavy or light chain V domain gene of rodent Ig derived proteinsobtained by phage display technique is replaced with a repertoire ofhuman V domain genes, creating rodent-human chimeras. Selection withantigen results in isolation of human variable domains capable ofrestoring a functional antigen-binding site, i.e. the epitope governs(imprints) the choice of partner. When the process is repeated in orderto replace the remaining rodent V domain, a human antibody is obtained(see PCT WO 93/06213, published 1 Apr. 1993). Unlike traditionalhumanization of rodent Ig derived proteins by CDR grafting, thistechnique provides completely human Ig derived proteins, which have noframework or CDR residues of rodent origin.

Bispecific Ig derived proteins can also be used that are monoclonal,preferably human or humanized, Ig derived proteins that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for at least one IL-23 protein; theother one is for any other antigen. For example, bispecific Ig derivedproteins specifically binding an IL-23 protein and at least oneneurotrophic factor, or two different types of IL-23 polypeptides arewithin the scope of the present invention.

Methods for making bispecific Ig derived proteins are known in the art.Traditionally, the recombinant production of bispecific Ig derivedproteins is based on the co-expression of two immunoglobulin heavychain-light chain pairs, where the two heavy chains have differentspecificities (Milstein and Cuello, Nature 305:537 (1983)). Because ofthe random assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in WO 93/08829published 13 May 1993, and in Traunecker et al., EMBO J. 10:3655 (1991),entirely incorporated herein by reference.

According to a different and more preferred approach, antibody-variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant-domain sequences.The fusion preferably is with an immunoglobulin heavy-chain constantdomain, comprising at least part of the hinge, the second heavy chainconstant region (C.sub.H 2), and the third heavy chain constant region(C.sub.H 3). It is preferred to have the first heavy-chain constantregion (C.sub.H 1), containing the site necessary for light-chainbinding, present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism. This provides for greatflexibility in adjusting the mutual proportions of the three polypeptidefragments in embodiments when unequal ratios of the three polypeptidechains used in the construction provide the optimum yields. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains in one expression vector when the production of atleast two polypeptide chains in equal ratios results in high yields orwhen the ratios are of no particular significance. In a preferredembodiment of this approach, the bispecific Ig derived proteins arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm.This asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. For furtherdetails of generating bispecific Ig derived proteins, see, for example,Suresh et al., Methods in Enzymology 121:210 (1986).

Heteroconjugate Ig derived proteins are also within the scope of thepresent invention. Heteroconjugate Ig derived proteins are composed oftwo covalently joined Ig derived proteins. Such Ig derived proteinshave, for example, been proposed to target immune system cells tounwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIVinfection (WO 91/00360; WO 92/00373; and EP 03089) Heteroconjugate Igderived proteins can be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

In a preferred embodiment, at least one anti-IL-23 Ig derived protein orspecified portion or variant of the present invention is produced by acell line, a mixed cell line, an immortalized cell or clonal populationof immortalized cells. Immortalized IL-23 producing cells can beproduced using suitable methods, for example, fusion of a human Igderived protein-producing cell and a heteromyeloma or immortalization ofan activated human B cell via infection with Epstein Barr virus(Niedbala et al., Hybridoma, 17(3):299-304 (1998); Zanella et al., JImmunol Methods, 156(2):205-215 (1992); Gustafsson et al., Hum Igderived proteins Hybridomas, 2(1)26-32 (1991)). Preferably, the humananti-human IL-23 proteins, fragments, specified portions or variants aregenerated by immunization of a transgenic animal (e.g., mouse, rat,hamster, non-human primate, and the like) capable of producing arepertoire of human Ig derived proteins, as described herein and/or asknown in the art. Cells that produce a human anti-IL-23 Ig derivedprotein can be isolated from such animals and immortalized usingsuitable methods, such as the methods described herein.

Transgenic mice that can produce a repertoire of human Ig derivedproteins that bind to human antigens can be produced by known methods(e.g., but not limited to, U.S. Pat. Nos. 5,770,428, 5,569,825,5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650issued to Lonberg et al.; Jakobovits et al. WO 98/50433, Jakobovits etal. WO 98/24893, Lonberg et al. WO 98/24884, Lonberg et al. WO 97/13852,Lonberg et al. WO 94/25585, Kucherlapate et al. WO 96/34096,Kucherlapate et al. EP 0463 151 B1, Kucherlapate et al. EP 0710 719 A1,Surani et al. U.S. Pat. No. 5,545,807, Bruggemann et al. WO 90/04036,Bruggemann et al. EP 0438 474 B1, Lonberg et al. EP 0814259 A2, Lonberget al. GB 2 272 440 A, Lonberg et al. Nature 368:856-859 (1994), Tayloret al., Int. Immunol. 6(4)579-591 (1994), Green et al., Nature Genetics7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Tayloret al., Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et al.,Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al., Int RevImmunol 13(1):65-93 (1995) and Fishwald et al., Nat Biotechnol14(7):845-851 (1996), which are each entirely incorporated herein byreference). Generally, these mice comprise at least one transgenecomprising DNA from at least one human immunoglobulin locus that isfunctionally rearranged, or which can undergo functional rearrangement.The endogenous immunoglobulin loci in such mice can be disrupted ordeleted to eliminate the capacity of the animal to produce Ig derivedproteins encoded by endogenous genes.

The term “functionally rearranged,” as used herein refers to a segmentof DNA from an immunoglobulin locus that has undergone V(D)Jrecombination, thereby producing an immunoglobulin gene that encodes animmunoglobulin chain (e.g., heavy chain, light chain), or any portionthereof. A functionally rearranged immunoglobulin gene can be directlyor indirectly identified using suitable methods, such as, for example,nucleotide sequencing, hybridization (e.g., Southern blotting, Northernblotting) using probes that can anneal to coding joints between genesegments or enzymatic amplification of immunoglobulin genes (e.g.,polymerase chain reaction) with primers that can anneal to coding jointsbetween gene segments. Whether a cell produces an Ig derived proteincomprising a particular variable region or a variable region comprisinga particular sequence (e.g., at least one CDR sequence) can also bedetermined using suitable methods. In one example, mRNA can be isolatedfrom an Ig derived protein-producing cell (e.g., a hybridoma orrecombinant cell or other suitable source) and used to produce cDNAencoding the Ig derived protein or specified portion or variant thereof.The cDNA can be cloned and sequenced or can be amplified (e.g., bypolymerase chain reactionor other known and suitable methods) using afirst primer that anneals specifically to a portion of the variableregion of interest (e.g., CDR, coding joint) and a second primer thatanneals specifically to non-variable region sequences (e.g., C_(H)1,V_(H)).

Screening Ig derived protein or specified portion or variants forspecific binding to similar proteins or fragments can be convenientlyachieved using peptide display libraries. This method involves thescreening of large collections of peptides for individual members havingthe desired function or structure. Ig derived protein screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 25amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT Patent Publication Nos.91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent Publication Nos. 92/05258,92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754; and5,643,768. Peptide display libraries, vector, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.), and Cambridge Ig derived protein Technologies (Cambridgeshire,UK). See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778,5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733,5,767,260, 5,856,456, assigned to Enzon; 5,223,409, 5,403,484,5,571,698, 5,837,500, assigned to Dyax, 5,427,908, 5,580,717, assignedto Affymax; 5,885,793, assigned to Cambridge Ig derived proteinTechnologies; 5,750,373, assigned to Genentech, 5,618,920, 5,595,898,5,576,195, 5,698,435, 5,693,493, 5,698,417, assigned to Xoma, Colligan,supra; Ausubel, supra; or Sambrook, supra, each of the above patents andpublications entirely incorporated herein by reference.

Ig derived proteins, specified portions and variants of the presentinvention can also be prepared by providing at least one IL-23 Igderived protein or specified portion or variant encoding nucleic acid totransgenic animals or mammals, such as goats, cows, horses, sheep, andthe like, that produce such Ig derived proteins or specified portions orvariants in their milk. Such animals can be provided using knownmethods. See, e.g., but not limited to, U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, andthe like, each of which is entirely incorporated herein by reference.

Ig derived proteins, specified portions and variants of the presentinvention can additionally be prepared using at least one IL-23 Igderived protein or specified portion or variant encoding nucleic acid toprovide transgenic plants and cultured plant cells (e.g., but notlimited to, tobacco and maize) that produce such Ig derived proteins,specified portions or variants in the plant parts or in cells culturedtherefrom. As a non-limiting example, transgenic tobacco leavesexpressing recombinant proteins have been successfully used to providelarge amounts of recombinant proteins, e.g., using an induciblepromoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol.240:95-118 (1999) and references cited therein. Also, transgenic maizehave been used to express mammalian proteins at commercial productionlevels, with biological activities equivalent to those produced in otherrecombinant systems or purified from natural sources. See, e.g., Hood etal., Adv. Exp. Med. Biol. 464:127-147 (1999) and references citedtherein. Ig derived proteins have also been produced in large amountsfrom transgenic plant seeds including Ig derived protein fragments, suchas single chain Ig derived proteins (scFv's), including tobacco seedsand potato tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109(1998) and reference cited therein. Thus, Ig derived proteins, specifiedportions and variants of the present invention can also be producedusing transgenic plants, according to known methods. See also, e.g.,Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October, 1999), Maet al., Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol.109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944(1994); and references cited therein. Each of the above references isentirely incorporated herein by reference.

The Ig derived proteins of the invention can bind human IL-23 proteins,receptors, and/or fragments with a wide range of affinities (K_(D)). Ina preferred embodiment, at least one human mAb of the present inventioncan optionally bind human IL-23 proteins, receptors, and/or fragmentswith high affinity. For example, a human mAb can bind human IL-23proteins, receptors, and/or fragments with a K_(D) equal to or less thanabout 10⁻⁹ M or, more preferably, with a K_(D) equal to or less thanabout 0.1-9.9 (or any range or value therein) X 10⁻¹⁰ M, 10⁻¹¹, 10⁻¹²,10⁻¹³ or any range or value therein.

The affinity or avidity of an Ig derived protein for an antigen can bedetermined experimentally using any suitable method. (See, for example,Berzofsky, et al., “Ig derived protein-Antigen Interactions,” InFundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y.(1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York,N.Y. (1992); and methods described herein). The measured affinity of aparticular Ig derived protein-antigen interaction can vary if measuredunder different conditions (e.g., salt concentration, pH). Thus,measurements of affinity and other antigen-binding parameters (e.g.,K_(D), K_(a), K_(d)) are preferably made with standardized solutions ofIg derived protein and antigen, and a standardized buffer, such as thebuffer described herein.

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 90-100% of the contiguous amino acids of at least oneIL-23 Ig derived protein of the present invention, specified fragments,variants or consensus sequences thereof, or a deposited vectorcomprising at least one of these sequences, a nucleic acid molecule ofthe present invention encoding at least one IL-23 Ig derived protein orspecified portion or variant can be obtained using methods describedherein or as known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain or light chain, respectively; nucleic acidmolecules comprising the coding sequence for an IL-23 Ig derived proteinor specified portion or variant; and nucleic acid molecules whichcomprise a nucleotide sequence substantially different from thosedescribed above but which, due to the degeneracy of the genetic code,still encode at least one IL-23 Ig derived protein as described hereinand/or as known in the art. Of course, the genetic code is well known inthe art. Thus, it would be routine for one skilled in the art togenerate such degenerate nucleic acid variants that code for specificIL-23 Ig derived protein or specified portion or variants of the presentinvention. See, e.g., Ausubel, et al., supra, and such nucleic acidvariants are included in the present invention.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an IL-23 Ig derived protein orspecified portion or variant can include, but are not limited to, thoseencoding the amino acid sequence of an Ig derived protein fragment, byitself; the coding sequence for the entire Ig derived protein or aportion thereof; the coding sequence for an Ig derived protein, fragmentor portion, as well as additional sequences, such as the coding sequenceof at least one signal leader or fusion peptide, with or without theaforementioned additional coding sequences, such as at least one intron,together with additional, non-coding sequences, including but notlimited to, non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals (forexample—ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding an Igderived protein or specified portion or variant can be fused to a markersequence, such as a sequence encoding a peptide that facilitatespurification of the fused Ig derived protein or specified portion orvariant comprising an Ig derived protein fragment or portion.

Polynucleotides Selectively Hybridizing to a Polynucleotide as DescribedHerein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide encoding aIL-23 Ig derived protein of the present invention. Thus, thepolynucleotides of this embodiment can be used for isolating, detecting,and/or quantifying nucleic acids comprising such polynucleotides. Forexample, polynucleotides of the present invention can be used toidentify, isolate, or amplify partial or full-length clones in adeposited library. In some embodiments, the polynucleotides are genomicor cDNA sequences isolated, or otherwise complementary to, a cDNA from ahuman or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an Ig derived protein or specified portion or variant encodedby the polynucleotides described herein. The polynucleotides of thisinvention embrace nucleic acid sequences that can be employed forselective hybridization to a polynucleotide encoding an Ig derivedprotein or specified portion or variant of the present invention. See,e.g., Ausubel, supra; Colligan, supra, each entirely incorporated hereinby reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, and (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent, such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 90-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 toInnis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 toGyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, etal; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediatedamplification that uses anti-sense RNA to the target sequence as atemplate for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek, et al., with the tradename NASBA), the entire contents of whichreferences are incorporated herein by reference. (See, e.g., Ausubel,supra; or Sambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene32 protein (Boehringer Mannheim) can be used to improve yield of longPCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example, a cDNA or a genomicsequence encoding an Ig derived protein or specified portion or variantof the present invention, can be used to construct a recombinantexpression cassette that can be introduced into at least one desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences that will direct thetranscription of the polynucleotide in the intended host cell. Bothheterologous and non-heterologous (i.e., endogenous) promoters can beemployed to direct expression of the nucleic acids of the presentinvention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

A polynucleotide of the present invention can be expressed in eithersense or anti-sense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable characteristics.

Another method of suppression is sense suppression. Introduction ofnucleic acid configured in the sense orientation has been shown to be aneffective means by which to block the transcription of target genes.

A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect and/or cleave nucleicacids. Knorre, et al., Biochimie 67:785-789 (1985); Vlassov, et al.,Nucleic Acids Res. 14:4065-4076 (1986); Iverson and Dervan, J. Am. Chem.Soc. 109:1241-1243 (1987); Meyer, et al., J. Am. Chem. Soc.111:8517-8519 (1989); Lee, et al., Biochemistry 27:3197-3203 (1988);Home, et al., J. Am. Chem. Soc. 112:2435-2437 (1990); Webb andMatteucci, J. Am. Chem. Soc. 108:2764-2765 (1986); Nucleic Acids Res.14:7661-7674 (1986); Feteritz, et al., J. Am. Chem. Soc. 113:4000(1991). Various compounds to bind, detect, label, and/or cleave nucleicacids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507;5,672,593; 5,484,908; 5,256,648; and 5,681,941, each entirelyincorporated herein by reference.

Vectors And Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one IL-23 Ig derived protein or specified portion or variantby recombinant techniques, as is well known in the art. See, e.g.,Sambrook, et al., supra; Ausubel, et al., supra, each entirelyincorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; and 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; and 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated herein by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one Ig derived protein or specified portion or variant of thepresent invention can be expressed in a modified form, such as a fusionprotein, and can include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, can be added to theN-terminus of an Ig derived protein or specified portion or variant toimprove stability and persistence in the host cell, during purification,or during subsequent handling and storage. Also, peptide moieties can beadded to an Ig derived protein or specified portion or variant of thepresent invention to facilitate purification. Such regions can beremoved prior to final preparation of an Ig derived protein or at leastone fragment thereof. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Alternatively, nucleic acids of the present invention can be expressedin a host cell by turning on (by manipulation) in a host cell thatcontains endogenous DNA encoding an Ig derived protein or specifiedportion or variant of the present invention. Such methods are well knownin the art, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670,5,733,746, and 5,733,761, entirely incorporated herein by reference.

Illustrative of cell cultures useful for the production of the Igderived proteins, specified portions or variants thereof, are mammaliancells. Mammalian cell systems often will be in the form of monolayers ofcells although mammalian cell suspensions or bioreactors can also beused. A number of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. Preferred host cells include cells of lymphoid origin, such asmyeloma and lymphoma cells. Particularly preferred host cells areP3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Ag14 cells(ATCC Accession Number CRL-1851). In a particularly preferredembodiment, the recombinant cell is a P3X63Ab8.653 or a SP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to, anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of an Ig Derived Protein or Specified Portion or VariantThereof

A IL-23 Ig derived protein or specified portion or variant can berecovered and purified from recombinant cell cultures by well-knownmethods, including, but not limited to, protein A purification, ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. High performance liquidchromatography (“HPLC”) can also be employed for purification. See e.g.,Colligan, Current Protocols in Immunology, or Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2003), e.g.,Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein byreference.

Ig derived proteins or specified portions or variants of the presentinvention include naturally purified products, products of chemicalsynthetic procedures, and products produced by recombinant techniquesfrom a eukaryotic host, including, for example, yeast, higher plant,insect and mammalian cells. Depending upon the host employed in arecombinant production procedure, the Ig derived protein or specifiedportion or variant of the present invention can be glycosylated or canbe non-glycosylated, with glycosylated preferred. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and20, Colligan, Protein Science, supra, Chapters 12-14, all entirelyincorporated herein by reference.

IL-23 Ig Derived Proteins, Fragments and/or Variants

The isolated Ig derived proteins of the present invention comprise an Igderived protein or specified portion or variant encoded by any one ofthe polynucleotides of the present invention as discussed more fullyherein, or any isolated or prepared Ig derived protein or specifiedportion or variant thereof.

Preferably, the human Ig derived protein or antigen-binding fragmentbinds human IL-23 proteins, receptors, and/or fragments and, therebysubstantially neutralizes the biological activity of the protein. An Igderived protein, or specified portion or variant thereof, that partiallyor preferably substantially neutralizes at least one biological activityof at least one IL-23 protein or fragment can bind the protein, receptorand/or fragment and thereby inhibit activities mediated through thebinding of IL-23 to at least one IL-23 receptor or through otherIL-23-dependent or mediated mechanisms. As used herein, the term“neutralizing Ig derived protein” refers to an Ig derived protein thatcan inhibit human p40 or p19 protein or fragment related-dependentactivity by about 20-120%, preferably by at least about 60, 70, 80, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay.The capacity of anti-human IL-23 Ig derived protein or specified portionor variant to inhibit human IL-23 related-dependent activity ispreferably assessed by at least one suitable IL-23 Ig derived protein orprotein assay, as described herein and/or as known in the art. A humanIg derived protein or specified portion or variant of the invention canbe of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and cancomprise a kappa or lambda light chain. In one embodiment, the human Igderived protein or specified portion or variant comprises an IgG heavychain or defined fragment, for example, at least one of isotypes, IgG1,IgG2, IgG3 or IgG4. Ig derived proteins of this type can be prepared byemploying a transgenic mouse or other trangenic non-human mammalcomprising at least one human light chain (e.g., IgG, IgA and IgM)(e.g., γ1, γ2, γ3, γ4) transgene as described herein and/or as known inthe art. In another embodiment, the anti-human IL-23 Ig derived proteinor specified portion or variant thereof comprises an IgG1 heavy chainand an IgG1 light chain.

At least one Ig derived protein or specified portion or variant of theinvention binds at least one specified epitope specific to at least oneIL-23 protein, receptor, subunit, fragment, portion or any combinationthereof. The at least one epitope can comprise at least one Ig derivedprotein binding region that comprises at least one portion of saidprotein, which epitope is preferably comprised of at least oneextracellular, soluble, hydrophillic, external or cytoplasmic portion ofsaid protein. As non-limiting examples, an IL-23 Ig derived protein orspecified portion or variant specifically binds at least one epitopecomprising (1) at least 1-3 amino acids of the entire amino acidsequence, selected from the group consisting of at least one p40 subunitof human IL-23, and (2) at least 1-3 amino acids of the entire aminoacid sequence of one or more IL-23 receptors. The at least one specifiedepitope can comprise any combination of at least one amino acid of thep40 subunit of a human interleukin-23, such as but not limited to, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids of at least oneof, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170,170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250,250-260, 260-270, 280-290, 290-300, 300-306, 1-7, 14-21, 29-52, 56-73,83-93, 96-105, 156-175, 194-204, 208-246, 254-273, 279-281, or 289-300of SEQ ID NO: 1 (human p40 subunit, 306 amino acids). Preferred examplesinclude 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the extracellulardomain of human IL-23p40 (SEQ ID NO:1), comprised of SEQ ID NO: 1. Morespecifically, the epitope regions are predicted to comprise Seg 1 (aminoacids 97-101 of SEQ ID NO:1), and/or Seg 2 (amino acids 138-145 of SEQID NO:1), and/or Seg 3 (amino acids 250-269 of SEQ ID NO:1) of SEQ IDNO:1.

These predicted epitopes are not available on the p40 subunit of IL-12,which is coupled to the p35 subunit of IL-12. These epitopes areavailable on the p40 subunit of IL-23, which is coupled to the p19subunit of IL-23. Accordingly, the antibody of the present inventionsubstantially does not react with IL-12 p40 and inhibits the interactionbetween IL-23 p40 and the IL-12Rβ1 receptor subunit

Generally, the human Ig derived protein or antigen-binding fragment ofthe present invention will comprise an antigen-binding region thatcomprises at least one human complementarity determining region (CDR1,CDR2 and CDR3) or variant of at least one heavy chain variable regionand at least one human complementarity determining region (CDR1, CDR2and CDR3) or variant of at least one light chain variable region. As anon-limiting example, the Ig derived protein or antigen-binding portionor variant can comprise at least one of the heavy chain CDR3, and/or alight chain CDR3. In a particular embodiment, the Ig derived protein orantigen-binding fragment can have an antigen-binding region thatcomprises at least a portion of at least one heavy chain CDR (i.e.,CDR1, CDR2 and/or CDR3) having the amino acid sequence of thecorresponding CDRs 1, 2 and/or 3. In another particular embodiment, theIg derived protein or antigen-binding portion or variant can have anantigen-binding region that comprises at least a portion of at least onelight chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acidsequence of the corresponding CDRs 1, 2 and/or 3. Such Ig derivedproteins can be prepared by chemically joining together the variousportions (e.g., CDRs, framework) of the Ig derived protein usingconventional techniques, by preparing and expressing a (i.e., one ormore) nucleic acid molecule that encodes the Ig derived protein usingconventional techniques of recombinant DNA technology or by using anyother suitable method.

The anti-IL-23 Ig derived protein can comprise at least one of a heavyor light chain variable region having a defined amino acid sequence. Forexample, in a preferred embodiment, the anti-IL-23 Ig derived proteincomprises at least one of at least one heavy chain variable regionand/or at least one light chain variable region. Human Ig derivedproteins that bind to human IL-23 proteins, receptors, and/or fragmentsand that comprise a defined heavy or light chain variable region can beprepared using suitable methods, such as phage display (Katsube, Y., etal., Int J Mol Med, 1(5):863-868 (1998)) or methods that employtransgenic animals, as known in the art and/or as described herein. Forexample, a transgenic mouse, comprising a functionally rearranged humanimmunoglobulin heavy chain transgene and a transgene comprising DNA froma human immunoglobulin light chain locus that can undergo functionalrearrangement, can be immunized with human IL-23 proteins, receptors,and/or fragments thereof to elicit the production of Ig derivedproteins. If desired, the Ig derived protein producing cells can beisolated and hybridomas or other immortalized Ig derivedprotein-producing cells can be prepared as described herein and/or asknown in the art. Alternatively, the Ig derived protein, specifiedportion or variant can be expressed using the encoding nucleic acid orportion thereof in a suitable host cell.

The invention also relates to Ig derived proteins, antigen-bindingfragments, immunoglobulin chains and CDRs comprising amino acids in asequence that is substantially the same as an amino acid sequencedescribed herein. Preferably, such Ig derived proteins orantigen-binding fragments and Ig derived proteins comprising such chainsor CDRs can bind human IL-23 proteins, receptors, and/or fragments withhigh affinity (e.g., K_(D) less than or equal to about 10⁻⁹ M). Aminoacid sequences that are substantially the same as the sequencesdescribed herein include sequences comprising conservative amino acidsubstitutions, as well as amino acid deletions and/or insertions. Aconservative amino acid substitution refers to the replacement of afirst amino acid by a second amino acid that has chemical and/orphysical properties (e.g., charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T.

Amino Acid Codes

The amino acids that make up IL-23 Ig derived proteins or specifiedportions or variants of the present invention are often abbreviated. Theamino acid designations can be indicated by designating the amino acidby its single letter code, its three letter code, name, or threenucleotide codon(s) as is well understood in the art (see Alberts, B.,et al., Molecular Biology of The Cell, Third Ed., Garland Publishing,Inc., New York, 1994):

SINGLE THREE THREE LETTER LETTER NUCLEOTIDE CODE CODE NAME CODON(S) AAla Alanine GCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D Asp Asparticacid GAC, GAU E Glu Glutamic acid GAA, GAG F Phe Phenylanine UUC, UUU GGly Glycine GGA, GGC, GGG, GGU H His Histidine CAC, CAU I Ile IsoleucineAUA, AUC, AUU K Lys Lysine AAA, AAG L Leu Leucine UUA, UUG, CUA, CUC,CUG, CUU M Met Methionine AUG N Asn Asparagine AAC, AAU P Pro ProlineCCA, CCC, CCG, CCU Q Gln Glutamine CAA, CAG R Arg Arginine AGA, AGG,CGA, CGC, CGG, CGU S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T ThrThreonine ACA, ACC, ACG, ACU V Val Valine GUA, GUC, GUG, GUU W TrpTryptophan UGG Y Tyr Tyrosine UAC, UAU

An IL-23 Ig derived protein or specified portion or variant of thepresent invention can include one or more amino acid substitutions,deletions or additions, either from natural mutations or humanmanipulation, as specified herein.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given IL-23 Ig derived polypeptide will not be morethan 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, 1, such as 1-30 or any range or value therein, as specifiedherein.

Amino acids in an IL-23 Ig derived protein or specified portion orvariant of the present invention that are essential for function can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra,Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity, such as, but not limited to, at least one IL-23neutralizing activity. Sites that are critical for Ig derived protein orspecified portion or variant binding can also be identified bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904(1992) and de Vos, et al., Science 255:306-312 (1992)).

The Ig derived proteins or specified portions or variants of the presentinvention, or specified variants thereof, can comprise any number ofcontiguous amino acid residues from an Ig derived protein or specifiedportion or variant of the present invention, wherein that number isselected from the group of integers consisting of from 10-100% of thenumber of contiguous residues in a IL-23 Ig derived protein or specifiedportion or variant. Optionally, this subsequence of contiguous aminoacids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 ormore amino acids in length, or any range or value therein. Further, thenumber of such subsequences can be any integer selected from the groupconsisting of from 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active Ig derived protein or specified portion orvariant of the present invention. Biologically active Ig derivedproteins or specified portions or variants have a specific activity atleast 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, andmost preferably at least 80%, 90%, or 95%-1000% of that of the native(non-synthetic), endogenous or related and known Ig derived protein orspecified portion or variant. Methods of assaying and quantifyingmeasures of enzymatic activity and substrate specificity, are well knownto those of skill in the art.

In another aspect, the invention relates to human Ig derived proteinsand antigen-binding fragments, as described herein, which are modifiedby the covalent attachment of an organic moiety.

Such modification can produce an Ig derived protein or antigen-bindingfragment with improved pharmacokinetic properties (e.g., increased invivo serum half-life). The organic moiety can be a linear or branchedhydrophilic polymeric group, fatty acid group, or fatty acid estergroup. In particular embodiments, the hydrophilic polymeric group canhave a molecular weight of about 800 to about 120,000 Daltons and can bea polyalkane glycol (e.g., polyethylene glycol (PEG), polypropyleneglycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinylpyrolidone, and the fatty acid or fatty acid ester group can comprisefrom about eight to about forty carbon atoms.

The modified Ig derived proteins and antigen-binding fragments of theinvention can comprise one or more organic moieties that are covalentlybonded, directly or indirectly, to the Ig derived protein or specifiedportion or variant. Each organic moiety that is bonded to an Ig derivedprotein or antigen-binding fragment of the invention can independentlybe a hydrophilic polymeric group, a fatty acid group or a fatty acidester group. As used herein, the term “fatty acid” encompassesmono-carboxylic acids and di-carboxylic acids. A “hydrophilic polymericgroup,” as the term is used herein, refers to an organic polymer that ismore soluble in water than in octane. For example, polylysine is moresoluble in water than in octane. Thus, an Ig derived protein modified bythe covalent attachment of polylysine is encompassed by the invention.Hydrophilic polymers suitable for modifying Ig derived proteins of theinvention can be linear or branched and include, for example, polyalkaneglycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and thelike), carbohydrates (e.g., dextran, cellulose, oligosaccharides,polysaccharides and the like), polymers of hydrophilic amino acids(e.g., polylysine, polyarginine, polyaspartate and the like), polyalkaneoxides (e.g., polyethylene oxide, polypropylene oxide and the like) andpolyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifiesthe Ig derived protein of the invention has a molecular weight of about800 to about 150,000 Daltons as a separate molecular entity. Forexample, PEG₅₀₀₀ and PEG_(20,000), wherein the subscript is the averagemolecular weight of the polymer in Daltons, can be used.

The hydrophilic polymeric group can be substituted with one to about sixalkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers thatare substituted with a fatty acid or fatty acid ester group can beprepared by employing suitable methods. For example, a polymercomprising an amine group can be coupled to a carboxylate of the fattyacid or fatty acid ester, and an activated carboxylate (e.g., activatedwith N,N-carbonyl diimidazole) on a fatty acid or fatty acid ester canbe coupled to a hydroxyl group on a polymer.

Fatty acids and fatty acid esters suitable for modifying Ig derivedproteins of the invention can be saturated or can contain one or moreunits of unsaturation. Fatty acids that are suitable for modifying Igderived proteins of the invention include, for example, n-dodecanoate(C₁₂, laurate), n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈,stearate), n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂,behenate), n-triacontanoate (C₃₀₋), n-tetracontanoate (C₄₀₋),cis-Δ9-octadecanoate (C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate(C₂₀, arachidonate), octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like. Suitable fattyacid esters include mono-esters of dicarboxylic acids that comprise alinear or branched lower alkyl group. The lower alkyl group can comprisefrom one to about twelve, preferably one to about six, carbon atoms.

The modified human Ig derived proteins and antigen-binding fragments canbe prepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups, such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example, adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom, such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221, the entire teachings of which areincorporated herein by reference.)

The modified Ig derived proteins of the invention can be produced byreacting a human Ig derived protein or antigen-binding fragment with amodifying agent. For example, the organic moieties can be bonded to theIg derived protein in a non-site specific manner by employing anamine-reactive modifying agent, for example, an NHS ester of PEG.Modified human Ig derived proteins or antigen-binding fragments can alsobe prepared by reducing disulfide bonds (e.g., intra-chain disulfidebonds) of an Ig derived protein or antigen-binding fragment. The reducedIg derived protein or antigen-binding fragment can then be reacted witha thiol-reactive modifying agent to produce the modified Ig derivedprotein of the invention. Modified human Ig derived proteins andantigen-binding fragments comprising an organic moiety that is bonded tospecific sites of an Ig derived protein or specified portion or variantof the present invention can be prepared using suitable methods, such asreverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992);Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al.,Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol Bioeng., 56(4):456-463(1997)), and the methods described in Hermanson, G. T., BioconjugateTechniques, Academic Press: San Diego, Calif. (1996).

Anti-Idiotype Antibodies to IL-23 Ig Derived Protein Compositions

In addition to IL-23 Ig derived proteins, the present invention is alsodirected to an anti-idiotypic (anti-Id) antibody specific for the IL-23Ig derived protein of the invention. An anti-Id antibody is an antibodythat recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal of the same species and genetic type (e.g.,mouse strain) as the source of the Id antibody with the antibody or aCDR containing region thereof. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing antibody andproduce an anti-Id antibody. The anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody.

The present invention also provides at least one IL-23 Ig derivedprotein composition comprising at least one, at least two, at leastthree, at least four, at least five, at least six or more anti-IL-1-23antibodies thereof, as described herein and/or as known in the art thatare provided in a non-naturally occurring composition, mixture or form.Such compositions comprise non-naturally occurring compositionscomprising at least one or two full length, C- and/or N-terminallydeleted variants, domains, fragments, or specified variants, of theIL-23 Ig derived protein amino acid sequence, or specified fragments,domains or variants thereof.

Antibody Compositions Comprising Further Therapeutically ActiveIngredients

The antibody compositions of the invention can optionally furthercomprise an effective amount of at least one compound or proteinselected from at least one of an anti-infective drug, a cardiovascular(CV) system drug, a central nervous system (CNS) drug, an autonomicnervous system (ANS) drug, a respiratory tract drug, a gastrointestinal(GI) tract drug, a hormonal drug, a drug for fluid or electrolytebalance, a hematologic drug, an antineoplastic, an immunomodulationdrug, an ophthalmic, otic or nasal drug, a topical drug, a nutritionaldrug or the like. Such drugs are well known in the art, includingformulations, indications, dosing and administration for each presentedherein (see, e.g., Nursing 2001 Handbook of Drugs, 21^(st) edition,Springhouse Corp., Springhouse, Pa., 2001; Health Professional's DrugGuide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, UpperSaddle River, N.J.; Pharmcotherapy Handbook, Wells et al., ed., Appleton& Lange, Stamford, Conn., each entirely incorporated herein byreference).

The anti-infective drug can be at least one selected from amebicides orat least one antiprotozoals, anthelmintics, antifungals, antimalarials,antituberculotics or at least one antileprotics, aminoglycosides,penicillins, cephalosporins, tetracyclines, sulfonamides,fluoroquinolones, antivirals, macrolide anti-infectives, andmiscellaneous anti-infectives. The CV drug can be at least one selectedfrom inotropics, antiarrhythmics, antianginals, antihypertensives,antilipemics, and miscellaneous cardiovascular drugs. The CNS drug canbe at least one selected from normarcotic analgesics or at least oneselected from antipyretics, nonsteroidal anti-inflammatory drugs,narcotic or at least one opiod analgesics, sedative-hypnotics,anticonvulsants, antidepressants, antianxiety drugs, antipsychotics,central nervous system stimulants, antiparkinsonians, and miscellaneouscentral nervous system drugs. The ANS drug can be at least one selectedfrom cholinergics (parasympathomimetics), anticholinergics, adrenergics(sympathomimetics), adrenergic blockers (sympatholytics), skeletalmuscle relaxants, and neuromuscular blockers. The respiratory tract drugcan be at least one selected from antihistamines, bronchodilators,expectorants or at least one antitussive, and miscellaneous respiratorydrugs. The GI tract drug can be at least one selected from antacids orat least one adsorbent or at least one antiflatulent, digestive enzymeor at least one gallstone solubilizer, antidiarrheals, laxatives,antiemetics, and antiulcer drugs. The hormonal drug can be at least oneselected from corticosteroids, androgens or at least one anabolicsteroid, estrogen or at least one progestin, gonadotropin, antidiabeticdrug or at least one glucagon, thyroid hormone, thyroid hormoneantagonist, pituitary hormone, and parathyroid-like drug. The drug forfluid and electrolyte balance can be at least one selected fromdiuretics, electrolytes or at least one replacement solution, acidifieror at least one alkalinizer. The hematologic drug can be at least oneselected from hematinics, anticoagulants, blood derivatives, andthrombolytic enzymes. The antineoplastics can be at least one selectedfrom alkylating drugs, antimetabolites, antibiotic antineoplastics,antineoplastics that alter hormone balance, and miscellaneousantineoplastics. The immunomodulation drug can be at least one selectedfrom immunosuppressants, vaccines or at least one toxoid, antitoxin orat least one antivenin, immune serum, and biological response modifier.The ophthalmic, otic, and nasal drugs can be at least one selected fromophthalmic anti-infectives, ophthalmic anti-inflammatories, miotics,mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics,otics, and nasal drugs. The topical drug can be at least one selectedfrom local anti-infectives, scabicides or at least one pediculicide ortopical corticosteroid. The nutritional drug can be at least oneselected from vitamins, minerals, or calorics. See, e.g., contents ofNursing 2001 Drug Handbook, supra.

The at least one amebicide or antiprotozoal can be at least one selectedfrom atovaquone, chloroquine hydrochloride, chloroquine phosphate,metronidazole, metronidazole hydrochloride, and pentamidine isethionate.The at least one anthelmintic can be at least one selected frommebendazole, pyrantel pamoate, and thiabendazole. The at least oneantifungal can be at least one selected from amphotericin B,amphotericin B cholesteryl sulfate complex, amphotericin B lipidcomplex, amphotericin B liposomal, fluconazole, flucytosine,griseofulvin microsize, griseofulvin ultramicrosize, itraconazole,ketoconazole, nystatin, and terbinafine hydrochloride. The at least oneantimalarial can be at least one selected from chloroquinehydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquinesulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine,and pyrimethamine with sulfadoxine. The at least one antituberculotic orantileprotic can be at least one selected from clofazimine, cycloserine,dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin,rifampin, rifapentine, and streptomycin sulfate. The at least oneaminoglycoside can be at least one selected from amikacin sulfate,gentamicin sulfate, neomycin sulfate, streptomycin sulfate, andtobramycin sulfate. The at least one penicillin can be at least oneselected from amoxcillin/clavulanate potassium, amoxicillin trihydrate,ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillinsodium/sulbactam sodium, cloxacillin sodium, dicloxacillin sodium,mezlocillin sodium, nafcillin sodium, oxacillin sodium, penicillin Gbenzathine, penicillin G potassium, penicillin G procaine, penicillin Gsodium, penicillin V potassium, piperacillin sodium, piperacillinsodium/tazobactam sodium, ticarcillin disodium, and ticarcillindisodium/clavulanate potassium. The at least one cephalosporin can be atleast one selected from cefaclor, cefadroxil, cefazolin sodium,cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium,cefonicid sodium, cefoperazone sodium, cefotaxime sodium, cefotetandisodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil,ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium,cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride,cephalexin monohydrate, cephradine, and loracarbef The at least onetetracycline can be at least one selected from demeclocyclinehydrochloride, doxycycline calcium, doxycycline hyclate, doxycyclinehydrochloride, doxycycline monohydrate, minocycline hydrochloride, andtetracycline hydrochloride. The at least one sulfonamide can be at leastone selected from co-trimoxazole, sulfadiazine, sulfamethoxazole,sulfisoxazole, and sulfisoxazole acetyl. The at least onefluoroquinolone can be at least one selected from alatrofloxacinmesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacinhydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, andtrovafloxacin mesylate. The at least one fluoroquinolone can be at leastone selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin,levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin,ofloxacin, sparfloxacin, and trovafloxacin mesylate. The at least oneantiviral can be at least one selected from abacavir sulfate, acyclovirsodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdinemesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium,foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine,lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivirphosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir,saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine,zanamivir, and zidovudine. The at least one macroline anti-infective canbe at least one selected from azithromycin, clarithromycin,dirithromycin, erythromycin base, erythromycin estolate, erythromycinethylsuccinate, erythromycin lactobionate, and erythromycin stearate.The at least one miscellaneous anti-infective can be at least oneselected from aztreonam, bacitracin, chloramphenicol sodium sucinate,clindamycin hydrochloride, clindamycin palmitate hydrochloride,clindamycin phosphate, imipenem and cilastatin sodium, meropenem,nitrofurantoin macrocrystals, nitrofurantoin microcrystals,quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim,and vancomycin hydrochloride. (See, e.g., pp. 24-214 of Nursing 2001Drug Handbook.)

The at least one inotropic can be at least one selected from aminonelactate, digoxin, and milrinone lactate. The at least one antiarrhythmiccan be at least one selected from adenosine, amiodarone hydrochloride,atropine sulfate, bretylium tosylate, diltiazem hydrochloride,disopyramide, disopyramide phosphate, esmolol hydrochloride, flecamideacetate, ibutilide fumarate, lidocaine hydrochloride, mexiletinehydrochloride, moricizine hydrochloride, phenyloin, phenyloin sodium,procainamide hydrochloride, propafenone hydrochloride, propranololhydrochloride, quinidine bisulfate, quinidine gluconate, quinidinepolygalacturonate, quinidine sulfate, sotalol, tocamide hydrochloride,and verapamil hydrochloride. The at least one antianginal can be atleast one selected from amlodipidine besylate, amyl nitrite, bepridilhydrochloride, diltiazem hydrochloride, isosorbide dinitrate, isosorbidemononitrate, nadolol, nicardipine hydrochloride, nifedipine,nitroglycerin, propranolol hydrochloride, verapamil, and verapamilhydrochloride. The at least one antihypertensive can be at least oneselected from acebutolol hydrochloride, amlodipine besylate, atenolol,benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate,candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol,clonidine, clonidine hydrochloride, diazoxide, diltiazem hydrochloride,doxazosin mesylate, enalaprilat, enalapril maleate, eprosartan mesylate,felodipine, fenoldopam mesylate, fosinopril sodium, guanabenz acetate,guanadrel sulfate, guanfacine hydrochloride, hydralazine hydrochloride,irbesartan, isradipine, labetalol hydrchloride, lisinopril, losartanpotassium, methyldopa, methyldopate hydrochloride, metoprolol succinate,metoprolol tartrate, minoxidil, moexipril hydrochloride, nadolol,nicardipine hydrochloride, nifedipine, nisoldipine, nitroprussidesodium, penbutolol sulfate, perindopril erbumine, phentolamine mesylate,pindolol, prazosin hydrochloride, propranolol hydrochloride, quinaprilhydrochloride, ramipril, telmisartan, terazosin hydrochloride, timololmaleate, trandolapril, valsartan, and verapamil hydrochloride. The atleast one antilipemic can be at least one selected from atorvastatincalcium, cerivastatin sodium, cholestyramine, colestipol hydrochloride,fenofibrate (micronized), fluvastatin sodium, gemfibrozil, lovastatin,niacin, pravastatin sodium, and simvastatin. The at least onemiscellaneous CV drug can be at least one selected from abciximab,alprostadil, arbutamine hydrochloride, cilostazol, clopidogrelbisulfate, dipyridamole, eptifibatide, midodrine hydrochloride,pentoxifylline, ticlopidine hydrochloride, and tirofiban hydrochloride.(See, e.g., pp. 215-336 of Nursing 2001 Drug Handbook.)

The at least one normarcotic analgesic or antipyretic can be at leastone selected from acetaminophen, aspirin, choline magnesiumtrisalicylate, diflunisal, and magnesium salicylate. The at least onenonsteroidal anti-inflammatory drug can be at least one selected fromcelecoxib, diclofenac potassium, diclofenac sodium, etodolac, fenoprofencalcium, flurbiprofen, ibuprofen, indomethacin, indomethacin sodiumtrihydrate, ketoprofen, ketorolac tromethamine, nabumetone, naproxen,naproxen sodium, oxaprozin, piroxicam, rofecoxib, and sulindac. The atleast one narcotic or opiod analgesic can be at least one selected fromalfentanil hydrochloride, buprenorphine hydrochloride, butorphanoltartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyltransdermal system, fentanyl transmucosal, hydromorphone hydrochloride,meperidine hydrochloride, methadone hydrochloride, morphinehydrochloride, morphine sulfate, morphine tartrate, nalbuphinehydrochloride, oxycodone hydrochloride, oxycodone pectinate, oxymorphonehydrochloride, pentazocine hydrochloride, pentazocine hydrochloride andnaloxone hydrochloride, pentazocine lactate, propoxyphene hydrochloride,propoxyphene napsylate, remifentanil hydrochloride, sufentanil citrate,and tramadol hydrochloride. The at least one sedative-hypnotic can be atleast one selected from chloral hydrate, estazolam, flurazepamhydrochloride, pentobarbital, pentobarbital sodium, phenobarbitalsodium, secobarbital sodium, temazepam, triazolam, zaleplon, andzolpidem tartrate. The at least one anticonvulsant can be at least oneselected from acetazolamide sodium, carbamazepine, clonazepam,clorazepate dipotassium, diazepam, divalproex sodium, ethosuximde,fosphenyloin sodium, gabapentin, lamotrigine, magnesium sulfate,phenobarbital, phenobarbital sodium, phenyloin, phenyloin sodium,phenyloin sodium (extended), primidone, tiagabine hydrochloride,topiramate, valproate sodium, and valproic acid. The at least oneantidepressant can be at least one selected from amitriptylinehydrochloride, amitriptyline pamoate, amoxapine, bupropionhydrochloride, citalopram hydrobromide, clomipramine hydrochloride,desipramine hydrochloride, doxepin hydrochloride, fluoxetinehydrochloride, imipramine hydrochloride, imipramine pamoate,mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride,paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride,tranylcypromine sulfate, trimipramine maleate, and venlafaxinehydrochloride. The at least one antianxiety drug can be at least oneselected from alprazolam, buspirone hydrochloride, chlordiazepoxide,chlordiazepoxide hydrochloride, clorazepate dipotassium, diazepam,doxepin hydrochloride, hydroxyzine embonate, hydroxyzine hydrochloride,hydroxyzine pamoate, lorazepam, mephrobamate, midazolam hydrochloride,and oxazepam. The at least one antipsychotic drug can be at least oneselected from chlorpromazine hydrochloride, clozapine, fluphenazinedecanoate, fluephenazine enanthate, fluphenazine hydrochloride,haloperidol, haloperidol decanoate, haloperidol lactate, loxapinehydrochloride, loxapine succinate, mesoridazine besylate, molindonehydrochloride, olanzapine, perphenazine, pimozide, prochlorperazine,quetiapine fumarate, risperidone, thioridazine hydrochloride,thiothixene, thiothixene hydrochloride, and trifluoperazinehydrochloride. The at least one central nervous system stimulant can beat least one selected from amphetamine sulfate, caffeine,dextroamphetamine sulfate, doxapram hydrochloride, methamphetaminehydrochloride, methylphenidate hydrochloride, modafinil, pemoline, andphentermine hydrochloride. The at least one antiparkinsonian can be atleast one selected from amantadine hydrochloride, benztropine mesylate,biperiden hydrochloride, biperiden lactate, bromocriptine mesylate,carbidopa-levodopa, entacapone, levodopa, pergolide mesylate,pramipexole dihydrochloride, ropinirole hydrochloride, selegilinehydrochloride, tolcapone, and trihexyphenidyl hydrochloride. The atleast one miscellaneous central nervous system drug can be at least oneselected from bupropion hydrochloride, donepezil hydrochloride,droperidol, fluvoxamine maleate, lithium carbonate, lithium citrate,naratriptan hydrochloride, nicotine polacrilex, nicotine transdermalsystem, propofol, rizatriptan benzoate, sibutramine hydrochloridemonohydrate, sumatriptan succinate, tacrine hydrochloride, andzolmitriptan. (See, e.g., pp. 337-530 of Nursing 2001 Drug Handbook.)

The at least one cholinergic (e.g., parasymathomimetic) can be at leastone selected from bethanechol chloride, edrophonium chloride,neostigmine bromide, neostigmine methylsulfate, physostigminesalicylate, and pyridostigmine bromide. The at least one anticholinergiccan be at least one selected from atropine sulfate, dicyclominehydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate,propantheline bromide, scopolamine, scopolamine butylbromide, andscopolamine hydrobromide. The at least one adrenergic (sympathomimetics)can be at least one selected from dobutamine hydrochloride, dopaminehydrochloride, metaraminol bitartrate, norepinephrine bitartrate,phenylephrine hydrochloride, pseudoephedrine hydrochloride, andpseudoephedrine sulfate. The at least one adrenergic blocker(sympatholytic) can be at least one selected from dihydroergotaminemesylate, ergotamine tartrate, methysergide maleate, and propranololhydrochloride. The at least one skeletal muscle relaxant can be at leastone selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprinehydrochloride, dantrolene sodium, methocarbamol, and tizanidinehydrochloride. The at least one neuromuscular blocker can be at leastone selected from atracurium besylate, cisatracurium besylate,doxacurium chloride, mivacurium chloride, pancuronium bromide,pipecuronium bromide, rapacuronium bromide, rocuronium bromide,succinylcholine chloride, tubocurarine chloride, and vecuronium bromide.(See, e.g., pp. 531-84 of Nursing 2001 Drug Handbook.)

The at least one antihistamine can be at least one selected frombrompheniramine maleate, cetirizine hydrochloride, chlorpheniraminemaleate, clemastine fumarate, cyproheptadine hydrochloride,diphenhydramine hydrochloride, fexofenadine hydrochloride, loratadine,promethazine hydrochloride, promethazine theoclate, and triprolidinehydrochloride. The at least one bronchodilator can be at least oneselected from albuterol, albuterol sulfate, aminophylline, atropinesulfate, ephedrine sulfate, epinephrine, epinephrine bitartrate,epinephrine hydrochloride, ipratropium bromide, isoproterenol,isoproterenol hydrochloride, isoproterenol sulfate, levalbuterolhydrochloride, metaproterenol sulfate, oxtriphylline, pirbuterolacetate, salmeterol xinafoate, terbutaline sulfate, and theophylline.The at least one expectorant or antitussive can be at least one selectedfrom benzonatate, codeine phosphate, codeine sulfate, dextramethorphanhydrobromide, diphenhydramine hydrochloride, guaifenesin, andhydromorphone hydrochloride. The at least one miscellaneous respiratorydrug can be at least one selected from acetylcysteine, beclomethasonedipropionate, beractant, budesonide, calfactant, cromolyn sodium, domasealfa, epoprostenol sodium, flunisolide, fluticasone propionate,montelukast sodium, nedocromil sodium, palivizumab, triamcinoloneacetonide, zafirlukast, and zileuton. (See, e.g., pp. 585-642 of Nursing2001 Drug Handbook.)

The at least one antacid, adsorbent, or antiflatulent can be at leastone selected from aluminum carbonate, aluminum hydroxide, calciumcarbonate, magaldrate, magnesium hydroxide, magnesium oxide,simethicone, and sodium bicarbonate. The at least one digestive enzymeor gallstone solubilizer can be at least one selected from pancreatin,pancrelipase, and ursodiol. The at least one antidiarrheal can be atleast one selected from attapulgite, bismuth subsalicylate, calciumpolycarbophil, diphenoxylate hydrochloride and atropine sulfate,loperamide, octreotide acetate, opium tincture, and opium tincure(camphorated). The at least one laxative can be at least one selectedfrom bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagradaaromatic fluidextract, cascara sagrada fluidextract, castor oil,docusate calcium, docusate sodium, glycerin, lactulose, magnesiumcitrate, magnesium hydroxide, magnesium sulfate, methylcellulose,mineral oil, polyethylene glycol or electrolyte solution, psyllium,senna, and sodium phosphates. The at least one antiemetic can be atleast one selected from chlorpromazine hydrochloride, dimenhydrinate,dolasetron mesylate, dronabinol, granisetron hydrochloride, meclizinehydrochloride, metocloproamide hydrochloride, ondansetron hydrochloride,perphenazine, prochlorperazine, prochlorperazine edisylate,prochlorperazine maleate, promethazine hydrochloride, scopolamine,thiethylperazine maleate, and trimethobenzamide hydrochloride. The atleast one antiulcer drug can be at least one selected from cimetidine,cimetidine hydrochloride, famotidine, lansoprazole, misoprostol,nizatidine, omeprazole, rabeprozole sodium, rantidine bismuth citrate,ranitidine hydrochloride, and sucralfate. (See, e.g., pp. 643-95 ofNursing 2001 Drug Handbook.)

The at least one coricosteroid can be at least one selected frombetamethasone, betamethasone acetate or betamethasone sodium phosphate,betamethasone sodium phosphate, cortisone acetate, dexamethasone,dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisoneacetate, hydrocortisone, hydrocortisone acetate, hydrocortisonecypionate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, prednisolone, prednisolone acetate,prednisolone sodium phosphate, prednisolone tebutate, prednisone,triamcino lone, triamcino lone acetonide, and triamcino lone diacetate.The at least one androgen or anabolic steroid can be at least oneselected from danazol, fluoxymesterone, methyltestosterone, nandrolonedecanoate, nandrolone phenpropionate, testosterone, testosteronecypionate, testosterone enanthate, testosterone propionate, andtestosterone transdermal system. The at least one estrogen or progestincan be at least one selected from esterified estrogens, estradiol,estradiol cypionate, estradiol/norethindrone acetate transdermal system,estradiol valerate, estrogens (conjugated), estropipate, ethinylestradiol, ethinyl estradiol and desogestrel, ethinyl estradiol andethynodiol diacetate, ethinyl estradiol and desogestrel, ethinylestradiol and ethynodiol diacetate, ethinyl estradiol andlevonorgestrel, ethinyl estradiol and norethindrone, ethinyl estradioland norethindrone acetate, ethinyl estradiol and norgestimate, ethinylestradiol and norgestrel, ethinyl estradiol and norethindrone andacetate and ferrous fumarate, levonorgestrel, medroxyprogesteroneacetate, mestranol and norethindron, norethindrone, norethindroneacetate, norgestrel, and progesterone. The at least one gonadroptropincan be at least one selected from ganirelix acetate, gonadorelineacetate, histrelin acetate, and menotropins. The at least oneantidiabetic or glucaon can be at least one selected from acarbose,chlorpropamide, glimepiride, glipizide, glucagon, glyburide, insulins,metformin hydrochloride, miglitol, pioglitazone hydrochloride,repaglinide, rosiglitazone maleate, and troglitazone. The at least onethyroid hormone can be at least one selected from levothyroxine sodium,liothyronine sodium, liotrix, and thyroid. The at least one thyroidhormone antagonist can be at least one selected from methimazole,potassium iodide, potassium iodide (saturated solution),propylthiouracil, radioactive iodine (sodium iodide ¹³¹I), and strongiodine solution. The at least one pituitary hormone can be at least oneselected from corticotropin, cosyntropin, desmophressin acetate,leuprolide acetate, repository corticotropin, somatrem, somatropin, andvasopressin. The at least one parathyroid-like drug can be at least oneselected from calcifediol, calcitonin (human), calcitonin (salmon),calcitriol, dihydrotachysterol, and etidronate disodium. (See, e.g., pp.696-796 of Nursing 2001 Drug Handbook.)

The at least one diuretic can be at least one selected fromacetazolamide, acetazolamide sodium, amiloride hydrochloride,bumetanide, chlorthalidone, ethacrynate sodium, ethacrynic acid,furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone,spironolactone, torsemide, triamterene, and urea. The at least oneelectrolyte or replacement solution can be at least one selected fromcalcium acetate, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, calciumlactate, calcium phosphate (dibasic), calcium phosphate (tribasic),dextran (high-molecular-weight), dextran (low-molecular-weight),hetastarch, magnesium chloride, magnesium sulfate, potassium acetate,potassium bicarbonate, potassium chloride, potassium gluconate, Ringer'sinjection, Ringer's injection (lactated), and sodium chloride. The atleast one acidifier or alkalinizer can be at least one selected fromsodium bicarbonate, sodium lactate, and tromethamine. (See, e.g., pp.797-833 of Nursing 2001 Drug Handbook.)

The at least one hematinic can be at least one selected from ferrousfumarate, ferrous gluconate, ferrous sulfate, ferrous sulfate (dried),iron dextran, iron sorbitol, polysaccharide-iron complex, and sodiumferric gluconate complex. The at least one anticoagulant can be at leastone selected from ardeparin sodium, dalteparin sodium, danaparoidsodium, enoxaparin sodium, heparin calcium, heparin sodium, and warfarinsodium. The at least one blood derivative can be at least one selectedfrom albumin 5%, albumin 25%, antihemophilic factor, anti-inhibitorcoagulant complex, antithrombin III (human), factor IX (human), factorIX complex, and plasma protein fractions. The at least one thrombolyticenzyme can be at least one selected from alteplase, anistreplase,reteplase (recombinant), streptokinase, and urokinase. (See, e.g., pp.834-66 of Nursing 2001 Drug Handbook.)

The at least one alkylating drug can be at least one selected frombusulfan, carboplatin, carmustine, chlorambucil, cisplatin,cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride,melphalan, melphalan hydrochloride, streptozocin, temozolomide, andthiotepa. The at least one antimetabolite can be at least one selectedfrom capecitabine, cladribine, cytarabine, floxuridine, fludarabinephosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate,methotrexate sodium, and thioguanine. The at least one antibioticantineoplastic can be at least one selected from bleomycin sulfate,dactinomycin, daunorubicin citrate liposomal, daunorubicinhydrochloride, doxorubicin hydrochloride, doxorubicin hydrochlorideliposomal, epirubicin hydrochloride, idarubicin hydrochloride,mitomycin, pentostatin, plicamycin, and valrubicin. The at least oneantineoplastic that alters hormone balance can be at least one selectedfrom anastrozole, bicalutamide, estramustine phosphate sodium,exemestane, flutamide, goserelin acetate, letrozole, leuprolide acetate,megestrol acetate, nilutamide, tamoxifen citrate, testolactone, andtoremifene citrate. The at least one miscellaneous antineoplastic can beat least one selected from asparaginase, bacillus Calmette-Guerin (BCG)(live intravesical), dacarbazine, docetaxel, etoposide, etoposidephosphate, gemcitabine hydrochloride, irinotecan hydrochloride,mitotane, mitoxantrone hydrochloride, paclitaxel, pegaspargase, porfimersodium, procarbazine hydrochloride, rituximab, teniposide, topotecanhydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristinesulfate, and vinorelbine tartrate. (See, e.g., pp. 867-963 of Nursing2001 Drug Handbook.)

The at least one immunosuppressant can be at least one selected fromazathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immuneglobulin, muromonab-CD3, mycophenolate mofetil, mycophenolate mofetilhydrochloride, sirolimus, and tacrolimus. The at least one vaccine ortoxoid can be at least one selected from BCG vaccine, cholera vaccine,diphtheria and tetanus toxoids (adsorbed), diphtheria and tetanustoxoids and acellular pertussis vaccine adsorbed, diphtheria and tetanustoxoids and whole-cell pertussis vaccine, Haemophilus b conjugatevaccines, hepatitis A vaccine (inactivated), hepatitis B vaccine(recombinant), influenza virus vaccine 1999-2000 trivalent types A & B(purified surface antigen), influenza virus vaccine 1999-2000 trivalenttypes A & B (subvirion or purified subvirion), influenza virus vaccine1999-2000 trivalent types A & B (whole virion), Japanese encephalitisvirus vaccine (inactivated), Lyme disease vaccine (recombinant OspA),measles and mumps and rubella virus vaccine (live), measles and mumpsand rubella virus vaccine (live attenuated), measles virus vaccine (liveattenuated), meningococcal polysaccharide vaccine, mumps virus vaccine(live), plague vaccine, pneumococcal vaccine (polyvalent), poliovirusvaccine (inactivated), poliovirus vaccine (live, oral, trivalent),rabies vaccine (adsorbed), rabies vaccine (human diploid cell), rubellaand mumps virus vaccine (live), rubella virus vaccine (live,attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid), typhoidvaccine (oral), typhoid vaccine (parenteral), typhoid Vi polysaccharidevaccine, varicella virus vaccine, and yellow fever vaccine. The at leastone antitoxin or antivenin can be at least one selected from black widowspider antivenin, Crotalidae antivenom (polyvalent), diphtheriaantitoxin (equine), and Micrurus fulvius antivenin. The at least oneimmune serum can be at least one selected from cytomegalovirus immuneglobulin (intraveneous), hepatitis B immune globulin (human), immuneglobulin intramuscular, immune globulin intravenous, rabies immuneglobulin (human), respiratory syncytial virus immune globulinintravenous (human), Rh₀(D) immune globulin (human), Rh₀(D) immuneglobulin intravenous (human), tetanus immune globulin (human), andvaricella-zoster immune globulin. The at least one biological responsemodifier can be at least one selected from aldesleukin, epoetin alfa,filgrastim, glatiramer acetate for injection, interferon alfacon-1,interferon alfa-2a (recombinant), interferon alfa-2b (recombinant),interferon beta-1a, interferon beta-1b (recombinant), interferongamma-1b, levamisole hydrochloride, oprelvekin, and sargramostim. (See,e.g., pp. 964-1040 of Nursing 2001 Drug Handbook.)

The at least one ophthalmic anti-infective can be selected formbacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin,gentamicin sulfate, ofloxacin 0.3%, polymyxin B sulfate, sulfacetamidesodium 10%, sulfacetamide sodium 15%, sulfacetamide sodium 30%,tobramycin, and vidarabine. The at least one ophthalmicanti-inflammatory can be at least one selected from dexamethasone,dexamethasone sodium phosphate, diclofenac sodium 0.1%, fluorometholone,flurbiprofen sodium, ketorolac tromethamine, prednisolone acetate(suspension) and prednisolone sodium phosphate (solution). The at leastone miotic can be at least one selected from acetylocholine chloride,carbachol (intraocular), carbachol (topical), echothiophate iodide,pilocarpine, pilocarpine hydrochloride, and pilocarpine nitrate. The atleast one mydriatic can be at least one selected from atropine sulfate,cyclopentolate hydrochloride, epinephrine hydrochloride, epinephrylborate, homatropine hydrobromide, phenylephrine hydrochloride,scopolamine hydrobromide, and tropicamide. The at least one ophthalmicvasoconstrictor can be at least one selected from naphazolinehydrochloride, oxymetazoline hydrochloride, and tetrahydrozolinehydrochloride. The at least one miscellaneous ophthalmic can be at leastone selected from apraclonidine hydrochloride, betaxolol hydrochloride,brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride,dorzolamide hydrochloride, emedastine difumarate, fluorescein sodium,ketotifen fumarate, latanoprost, levobunolol hydrochloride, metipranololhydrochloride, sodium chloride (hypertonic), and timolol maleate. The atleast one otic can be at least one selected from boric acid, carbamideperoxide, chloramphenicol, and triethanolamine polypeptideoleate-condensate. The at least one nasal drug can be at least oneselected from beclomethasone dipropionate, budesonide, ephedrinesulfate, epinephrine hydrochloride, flunisolide, fluticasone propionate,naphazoline hydrochloride, oxymetazoline hydrochloride, phenylephrinehydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide,and xylometazoline hydrochloride. (See, e.g., pp. 1041-97 of Nursing2001 Drug Handbook.)

The at least one local anti-infective can be at least one selected fromacyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazolenitrate, clindamycin phosphate, clotrimazole, econazole nitrate,erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate,metronidazole (topical), miconazole nitrate, mupirocin, naftifinehydrochloride, neomycin sulfate, nitrofurazone, nystatin, silversulfadiazine, terbinafine hydrochloride, terconazole, tetracyclinehydrochloride, tioconazole, and tolnaftate. The at least one scabicideor pediculicide can be at least one selected from crotamiton, lindane,permethrin, and pyrethrins. The at least one topical corticosteroid canbe at least one selected from betamethasone dipropionate, betamethasonevalerate, clobetasol propionate, desonide, desoximetasone,dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate,fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasonepropionate, halcionide, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocorisone valerate, mometasone furoate, andtriamcinolone acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001 DrugHandbook.)

The at least one vitamin or mineral can be at least one selected fromvitamin A, vitamin B complex, cyanocobalamin, folic acid,hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxinehydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D,cholecalciferol, ergocalciferol, vitamin D analogue, doxercalciferol,paricalcitol, vitamin E, vitamin K analogue, phytonadione, sodiumfluoride, sodium fluoride (topical), trace elements, chromium, copper,iodine, manganese, selenium, and zinc. The at least one caloric can beat least one selected from amino acid infusions (crystalline), aminoacid infusions in dextrose, amino acid infusions with electrolytes,amino acid infusions with electrolytes in dextrose, amino acid infusionsfor hepatic failure, amino acid infusions for high metabolic stress,amino acid infusions for renal failure, dextrose, fat emulsions, andmedium-chain triglycerides. (See, e.g., pp. 1137-63 of Nursing 2001 DrugHandbook.)

IL-23 Ig Derived Protein or Specified Portion or Variant Compositions

The present invention also provides at least one IL-23 Ig derivedprotein or specified portion or variant composition comprising at leastone, at least two, at least three, at least four, at least five, atleast six or more IL-23 Ig derived proteins or specified portions orvariants thereof, as described herein and/or as known in the art thatare provided in a non-naturally occurring composition, mixture or form.Such compositions comprise non-naturally occurring compositionscomprising at least one or two full length, C- and/or N-terminallydeleted variants, domains, fragments, or specified variants, of theIL-23 Ig derived protein amino acid sequence, or specified fragments,domains or variants thereof. Such composition percentages are by weight,volume, concentration, molarity, or molality as liquid or dry solutions,mixtures, suspension, emulsions or colloids, as known in the art or asdescribed herein.

IL-23 Ig derived protein or specified portion or variant compositions ofthe present invention can further comprise at least one of any suitableauxiliary, such as, but not limited to, diluent, binder, stabilizer,buffers, salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the IL-23 composition as well known in the art or asdescribed herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin, such ashuman serum albumin (HSA), recombinant human albumin (rHA), gelatin,casein, and the like. Representative amino acid/Ig derived protein orspecified portion or variant components, which can also function in abuffering capacity, include alanine, glycine, arginine, betaine,histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine,isoleucine, valine, methionine, phenylalanine, aspartame, and the like.One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

IL-23 Ig derived protein compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid salts,such as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts, such as citrate.

Additionally, the IL-23 Ig derived protein or specified portion orvariant compositions of the invention can include polymericexcipients/additives, such as polyvinylpyrrolidones, ficolls (apolymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the IL-23 Ig protein compositions according to theinvention are known in the art, e.g., as listed in “Remington: TheScience & Practice of Pharmacy”, 19^(th) ed., Williams & Williams,(1995), and in the “Physician's Desk Reference”, 52^(nd) ed., MedicalEconomics, Montvale, N.J. (1998), the disclosures of which are entirelyincorporated herein by reference. Preferred carrier or excipientmaterials are carbohydrates (e.g., saccharides and alditols) and buffers(e.g., citrate) or polymeric agents.

Formulations

As noted above, the invention provides for stable formulations, whichcomprise preferably a phosphate buffer with saline or a chosen salt, aswell as preserved solutions and formulations containing a preservative,as well as multi-use preserved formulations suitable for pharmaceuticalor veterinary use, comprising at least one IL-23 Ig derived protein orspecified portion or variant in a pharmaceutically acceptableformulation. Preserved formulations contain at least one knownpreservative or optionally selected from the group consisting of atleast one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzylalcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde,chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,benzethonium chloride, sodium dehydroacetate and thimerosal, or mixturesthereof in an aqueous diluent. Any suitable concentration or mixture canbe used as known in the art, such as 0.001-5%, or any range or valuetherein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01,0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or valuetherein. Non-limiting examples include, no preservative, 0.1-2% m-cresol(e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5,0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005,0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%),0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005,0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75,0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one IL-23 Ig derived protein or specified portionor variant with the prescribed buffers and/or preservatives, optionally,in an aqueous diluent, wherein said packaging material comprises a labelthat indicates that such solution can be held over a period of 1, 2, 3,4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours orgreater. The invention further comprises an article of manufacture,comprising packaging material, a first vial comprising in lyophilizedform at least one IL-23 Ig derived protein or specified portion orvariant, and a second vial comprising an aqueous diluent of prescribedbuffer or preservative, wherein said packaging material comprises alabel that instructs a patient to reconstitute the at least one IL-23 Igderived protein or specified portion or variant in the aqueous diluentto form a solution that can be held over a period of twenty-four hoursor greater.

The at least one IL-23 Ig derived protein or specified portion orvariant used in accordance with the present invention can be produced byrecombinant means, including from mammalian cell or transgenicpreparations, or can be purified from other biological sources, asdescribed herein or as known in the art.

The range of at least one IL-23 Ig derived protein or specified portionor variant in the product of the present invention includes amountsyielding upon reconstitution, if in a wet/dry system, concentrationsfrom about 1.0 pg/ml to about 1000 mg/ml, although lower and higherconcentrations are operable and are dependent on the intended deliveryvehicle, e.g., solution formulations will differ from transdermal patch,pulmonary, transmucosal, or osmotic or micro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably, the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably, sodium phosphate,particularly, phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants, such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators, such asEDTA and EGTA, can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one IL-23 Ig derived protein orspecified portion or variant and a preservative selected from the groupconsisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzylalcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like),benzalkonium chloride, benzethonium chloride, sodium dehydroacetate andthimerosal or mixtures thereof in an aqueous diluent. Mixing the atleast one IL-23 Ig derived protein or specified portion or variant andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one IL-23 Ig derived proteinor specified portion or variant in buffered solution is combined withthe desired preservative in a buffered solution in quantities sufficientto provide the protein and preservative at the desired concentrations.Variations of this process would be recognized by one of ordinary skillin the art. For example, the order the components are added, whetheradditional additives are used, the temperature and pH at which theformulation is prepared, are all factors that may be optimized for theconcentration and means of administration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized, at least one IL-23 Igderived protein or specified portion or variant that is reconstitutedwith a second vial containing water, a preservative and/or excipients,preferably, a phosphate buffer and/or saline and a chosen salt, in anaqueous diluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus can provide a moreconvenient treatment regimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period ranging from immediately to twenty-fourhours or greater. Accordingly, the presently claimed articles ofmanufacture offer significant advantages to the patient. Formulations ofthe invention can optionally be safely stored at temperatures of fromabout 2 to about 40° C. and retain the biologically activity of theprotein for extended periods of time, thus allowing a package labelindicating that the solution can be held and/or used over a period of 6,12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent isused, such label can include use up to 1-12 months, one-half, one and ahalf, and/or two years.

The solutions of at least one IL-23 Ig derived protein or specifiedportion or variant in the invention can be prepared by a process thatcomprises mixing at least one Ig derived protein or specified portion orvariant in an aqueous diluent. Mixing is carried out using conventionaldissolution and mixing procedures. To prepare a suitable diluent, forexample, a measured amount of at least one Ig derived protein orspecified portion or variant in water or buffer is combined inquantities sufficient to provide the protein and, optionally, apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order in which the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that may be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized, at least one IL-23 Igderived protein or specified portion or variant that is reconstitutedwith a second vial containing the aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus provides a more convenient treatment regimenthan currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized, at least oneIL-23 Ig derived protein or specified portion or variant that isreconstituted with a second vial containing the aqueous diluent. Theclear solution in this case can be up to one liter or even larger insize, providing a large reservoir from which smaller portions of the atleast one Ig derived protein or specified portion or variant solutioncan be retrieved one or multiple times for transfer into smaller vialsand provided by the pharmacy or clinic to their customers and/orpatients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution, such as BD Pens, BDAutojecto®, Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®,Biojector®, Iject®, J-tip Needle-Free injector®, Intraject®, Medi-Ject®,e.g., as made or developed by Becton Dickenson (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com). Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution, such as theHumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one IL-23 Ig derived protein orspecified portion or variant in the aqueous diluent to form a solutionand to use the solution over a period of 2-24 hours or greater for thetwo vial, wet/dry, product. For the single vial, solution product, thelabel indicates that such solution can be used over a period of 2-24hours or greater. The presently claimed products are useful for humanpharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one IL-23 Ig derived protein or specifiedportion or variant and a selected buffer, preferably, a phosphate buffercontaining saline or a chosen salt. Mixing the at least one Ig derivedprotein or specified portion or variant and buffer in an aqueous diluentis carried out using conventional dissolution and mixing procedures. Toprepare a suitable formulation, for example, a measured amount of atleast one Ig derived protein or specified portion or variant in water orbuffer is combined with the desired buffering agent in water inquantities sufficient to provide the protein and buffer at the desiredconcentrations. Variations of this process would be recognized by one ofordinary skill in the art. For example, the order in which thecomponents are added, whether additional additives are used, thetemperature and pH at which the formulation is prepared, are all factorsthat can be optimized for the concentration and means of administrationused.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized, atleast one IL-23 Ig derived protein or specified portion or variant thatis reconstituted with a second vial containing a preservative or bufferand excipients in an aqueous diluent. Either a single solution vial ordual vial requiring reconstitution can be reused multiple times and cansuffice for a single or multiple cycles of patient treatment and thusprovides a more convenient treatment regimen than currently available.

At least one IL-23 Ig derived protein or specified portion or variant ineither the stable or preserved formulations or solutions describedherein, can be administered to a patient in accordance with the presentinvention via a variety of delivery methods including SC or IMinjection; transdermal, pulmonary, transmucosal, implant, osmotic pump,cartridge, micro pump, or other means appreciated by the skilledartisan, as well-known in the art.

Therapeutic Applications

The present invention also provides a method for modulating or treatingIL-23 conditions or diseases, in a cell, tissue, organ, animal, orpatient including, but not limited to, at least one of rheumatoidarthritis, juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis,gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatorybowel disease, ulcerative colitis, systemic lupus erythematosis,antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis,idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever,perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria,systemic anaphalaxis, dermatitis, pernicious anemia, hemolyticdisesease, thrombocytopenia, graft rejection of any organ or tissue,kidney transplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalmic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to, asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, acute or chronic bacterialinfection, acute and chronic parasitic or infectious processes,including bacterial, viral and fungal infections, HIV infection/HIVneuropathy, meningitis, hepatitis (e.g., A, B or C, or the like), septicarthritis, peritonitis, pneumonia, epiglottitis, e. Coli 0157:h7,hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shocksyndrome, streptococcal myositis, gas gangrene, mycobacteriumtuberculosis, mycobacterium avium intracellulare, pneumocystis cariniipneumonia, pelvic inflammatory disease, orchitis/epidydimitis,legionella, lyme disease, influenza a, epstein-barr virus,vital-associated hemaphagocytic syndrome, vital encephalitis/asepticmeningitis, neurodegenerative diseases, multiple sclerosis, migraineheadache, AIDS dementia complex, demyelinating diseases, such asmultiple sclerosis and acute transverse myelitis; extrapyramidal andcerebellar disorders, such as lesions of the corticospinal system;disorders of the basal ganglia or cerebellar disorders; hyperkineticmovement disorders, such as Huntington's Chorea and senile chorea;drug-induced movement disorders, such as those induced by drugs whichblock CNS dopamine receptors; hypokinetic movement disorders, such asParkinson's disease; Progressive supranucleo Palsy; structural lesionsof the cerebellum; spinocerebellar degenerations, such as spinal ataxia,Friedreich's ataxia, cerebellar cortical degenerations, multiple systemsdegenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph);systemic disorders (Refsum's disease, abetalipoprotemia, ataxia,telangiectasia, and mitochondrial multi-system disorder); demyelinatingcore disorders, such as multiple sclerosis, acute transverse myelitis;and disorders of the motor unit, such as neurogenic muscular atrophies(anterior horn cell degeneration, such as amyotrophic lateral sclerosis,infantile spinal muscular atrophy and juvenile spinal muscular atrophy);Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy bodydisease; Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome;chronic alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,neurotraumatic injury (e.g., but not limited to, spinal cord injury,brain injury, concussion, and repetitive concussion), pain, inflammatorypain, autism, depression, stroke, cognitive disorders, epilepsy, and thelike. Such a method can optionally comprise administering an effectiveamount of at least one composition or pharmaceutical compositioncomprising at least one IL-23 Ig derived protein or specified portion orvariant to a cell, tissue, organ, animal or patient in need of suchmodulation, treatment or therapy.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one IL-23 Ig derived protein or specified portion orvariant to a cell, tissue, organ, animal or patient in need of suchmodulation, treatment or therapy. Such a method can optionally furthercomprise co-administration or combination therapy for treating suchimmune diseases, wherein the administering of said at least one IL-23 Igderived protein, specified portion or variant thereof, further comprisesadministering, before, concurrently, and/or after, at least one selectedfrom at least one multiple sclerosis therapeutic (including but notlimited to, beta-interferon 1a and beta-interferon 1b (e.g., Avonex™,RebifrM™, Betaseon™), glutiramer acetate (e.g., Copaxone),cyclophasphamide, azathioprine, glucocorticosteroids, methotrexate,Paclitaxel, 2-chlorodeoxyadenosine, mitoxantrone, IL-10, TGBb, CD4,CD52, antegren, CD11, CD18, TNFalpha, IL-1, IL-2, and/or CD4 antibody orantibody receptor fusion, interferon alpha, immunoglobulin, Lismide(Requinimax™), insulin-like growth factor-1 (IGF-1), elprodil,pirfenidone, oral myelin, or compounds that act on one or more of atleast one of: autoimmune suppression of myelin destruction, immuneregulation, activation, proliferation, migration and/or suppressor cellfunction of T-cells, inhibition of T cell receptor/peptide/MHC-11interaction, induction of T cell anergy, deletion of autoreactive Tcells, reduction of trafficking across blood brain barrier, alterationof balance of pro-inflammatory (Th1) and immunomodulatory (Th2)cytokines, inhibition of matrix metalloprotease inhibitors,neuroprotection, reduction of gliosis, promotion of re-myelination), TNFantagonist (e.g., but not limited to a TNF Ig derived protein orfragment, a soluble TNF receptor or fragment, fusion proteins thereof,or a small molecule TNF antagonist), an antirheumatic, a musclerelaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), ananalgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a fluororquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, an IL-23 agent, a mineral, anutritional, a thyroid agent, a vitamin, a calcium related hormone, anantidiarrheal, an antitussive, an antiemetic, an antiulcer, a laxative,an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim(e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one antibody, specified portion and variantthereof, of the present invention), include, but are not limited to,anti-TNF Ig derived proteins, antigen-binding fragments thereof, andreceptor molecules which bind specifically to TNF; compounds whichprevent and/or inhibit TNF synthesis, TNF release or its action ontarget cells, such as thalidomide, tenidap, phosphodiesterase inhibitors(e.g., pentoxifylline and rolipram), A2b adenosine receptor agonists andA2b adenosine receptor enhancers; compounds which prevent and/or inhibitTNF receptor signalling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

As used herein, a “tumor necrosis factor Ig derived protein,” “TNF Igderived protein,” “TNFα Ig derived protein,” or fragment and the likedecreases, blocks, inhibits, abrogates or interferes with TNFα activityin vitro, in situ and/or preferably in vivo. For example, a suitable TNFhuman Ig derived protein of the present invention can bind TNFα andincludes anti-TNF Ig derived proteins, antigen-binding fragmentsthereof, and specified mutants or domains thereof that bind specificallyto TNFα. A suitable TNF antibody or fragment can also decrease block,abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or proteinsynthesis, TNF release, TNF receptor signaling, membrane TNF cleavage,TNF activity, TNF production and/or synthesis.

Chimeric Ig derived protein cA2 consists of the antigen binding variableregion of the high-affinity neutralizing mouse anti-human TNFα IgG 1 Igderived protein, designated A2, and the constant regions of a humanIgG1, kappa immunoglobulin. The human IgG1 Fc region improves allogeneicIg derived protein effector function, increases the circulating serumhalf-life and decreases the immunogenicity of the Ig derived protein.The avidity and epitope specificity of the chimeric Ig derived proteincA2 is derived from the variable region of the murine Ig derived proteinA2. In a particular embodiment, a preferred source for nucleic acidsencoding the variable region of the murine Ig derived protein A2 is theA2 hybridoma cell line.

Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural andrecombinant human TNFα in a dose dependent manner. From binding assaysof chimeric Ig derived protein cA2 and recombinant human TNFα, theaffinity constant of chimeric Ig derived protein cA2 was calculated tobe 1.04×10¹⁰M⁻¹. Preferred methods for determining monoclonal Ig derivedprotein specificity and affinity by competitive inhibition can be foundin Harlow, et al., Ig derived proteins: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988; Colligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience, New York, (1992-2003); Kozbor et al., Immunol.Today, 4:72-79 (1983); Ausubel et al., eds. Current Protocols inMolecular Biology, Wiley Interscience, New York (1987-2003); and Muller,Meth. Enzymol., 92:589-601 (1983), which references are entirelyincorporated herein by reference.

In a particular embodiment, murine monoclonal Ig derived protein A2 isproduced by a cell line designated c134A. Chimeric Ig derived proteincA2 is produced by a cell line designated c168A.

Additional examples of monoclonal anti-TNF Ig derived proteins that canbe used in the present invention are described in the art (see, e.g.,U.S. Pat. No. 5,231,024; Möller, A. et al., Cytokine 2(3):162-169(1990); U.S. application Ser. No. 07/943,852 (filed Sep. 11, 1992);Rathjen et al., International Publication No. WO 91/02078 (publishedFeb. 21, 1991); Rubin et al., EPO Patent Publication No. 0 218 868(published Apr. 22, 1987); Yone et al., EPO Patent Publication No. 0 288088 (Oct. 26, 1988); Liang, et al., Biochem. Biophys. Res. Comm.137:847-854 (1986); Meager, et al., Hybridoma 6:305-311 (1987); Fendlyet al., Hybridoma 6:359-369 (1987); Bringman, et al., Hybridoma6:489-507 (1987); and Hirai, et al., J. Immunol. Meth. 96:57-62 (1987),which references are entirely incorporated herein by reference).

TNF Receptor Molecules

Preferred TNF receptor molecules useful in the present invention arethose that bind TNFα with high affinity (see, e.g., Feldmann et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992);Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell61:351-359 (1990), which references are entirely incorporated herein byreference) and optionally possess low immunogenicity. In particular, the55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptorsare useful in the present invention. Truncated forms of these receptors,comprising the extracellular domains (ECD) of the receptors orfunctional portions thereof (see, e.g., Corcoran et al., Eur. J.Biochem. 223:831-840 (1994)), are also useful in the present invention.Truncated forms of the TNF receptors, comprising the ECD, have beendetected in urine and serum as 30 kDa and 40 kDa TNFα inhibitory bindingproteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)).TNF receptor multimeric molecules and TNF immunoreceptor fusionmolecules, and derivatives and fragments or portions thereof, areadditional examples of TNF receptor molecules which are useful in themethods and compositions of the present invention. The TNF receptormolecules which can be used in the invention are characterized by theirability to treat patients for extended periods with good to excellentalleviation of symptoms and low toxicity. Low immunogenicity and/or highaffinity, as well as other undefined properties, may contribute to thetherapeutic results achieved.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). The multimeric molecules canfurther comprise a signal peptide of a secreted protein to directexpression of the multimeric molecule. These multimeric molecules andmethods for their production have been described in U.S. applicationSer. No. 08/437,533 (filed May 9, 1995), the content of which isentirely incorporated herein by reference.

TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein. TNF immunoreceptor fusion molecules and methods for theirproduction have been described in the art (Lesslauer et al., Eur. J.Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J.Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851;and U.S. application Ser. No. 08/442,133 (filed May 16, 1995), each ofwhich references are entirely incorporated herein by reference). Methodsfor producing immunoreceptor fusion molecules can also be found in Caponet al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538;and Capon et al., Nature 337:525-531 (1989), which references areentirely incorporated herein by reference.

A functional equivalent, derivative, fragment or region of TNF receptormolecule refers to the portion of the TNF receptor molecule, or theportion of the TNF receptor molecule sequence which encodes TNF receptormolecule, that is of sufficient size and sequences to functionallyresemble TNF receptor molecules that can be used in the presentinvention (e.g., bind TNFα with high affinity and possess lowimmunogenicity). A functional equivalent of TNF receptor molecule alsoincludes modified TNF receptor molecules that functionally resemble TNFreceptor molecules that can be used in the present invention (e.g., bindTNFα with high affinity and possess low immunogenicity). For example, afunctional equivalent of TNF receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2003).

Cytokines include any known cytokine. See, e.g., CopewithCytokines.com.Cytokine antagonists include, but are not limited to, any Ig derivedprotein, fragment or mimetic, any soluble receptor, fragment or mimetic,any small molecule antagonist, or any combination thereof.

Therapeutic Treatments

Any method of the present invention can comprise a method for treating aIL-23 mediated disorder, comprising administering an effective amount ofa composition or pharmaceutical composition comprising at least oneIL-23 Ig derived protein or specified portion or variant to a cell,tissue, organ, animal or patient in need of such modulation, treatmentor therapy.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one IL-23 Igrelated protein composition that total, on average, a range from atleast about 0.01 to 500 milligrams of at least one IL-23 Ig derivedprotein or specified portion or variant/kilogram of patient per dose,and, preferably, from at least about 0.1 to 100 milligrams of Ig derivedprotein or specified portion or variant/kilogram of patient per singleor multiple administration, depending upon the specific activity ofcontained in the composition. Alternatively, the effective serumconcentration can comprise 0.1-5000 μg/ml serum concentration per singleor multiple administration. Suitable dosages are known to medicalpractitioners and will, of course, depend upon the particular diseasestate, specific activity of the composition being administered, and theparticular patient undergoing treatment. In some instances, to achievethe desired therapeutic amount, it can be necessary to provide forrepeated administration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100 mg/kg/administration,or any range, value or fraction thereof, or to achieve a serumconcentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9,3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5,7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20,12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15.9,16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20,20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000 μg/mlserum concentration per single or multiple administration, or any range,value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually, adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily, 0.1 to 50, and, preferably, 0.1 to10 milligrams per kilogram per administration or in sustained releaseform is effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one Ig derivedprotein or specified portion or variant of the present invention, 0.1 to100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at leastone of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or 40, or, alternatively or additionally, at least oneof week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, or,alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or anycombination thereof, using single, infusion or repeated doses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions, the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the Ig derived protein or specifiedportion or variant can be formulated as a solution, suspension, emulsionor lyophilized powder in association, or separately provided, with apharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and1-10% human serum albumin. Liposomes and nonaqueous vehicles, such asfixed oils, may also be used. The vehicle or lyophilized powder maycontain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by known or suitable techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration

Many known and developed modes can be used according to the presentinvention for administering pharmaceutically effective amounts of atleast one IL-23 Ig derived protein or specified portion or variantaccording to the present invention. While pulmonary administration isused in the following description, other modes of administration can beused according to the present invention with suitable results.

IL-23 Ig derived proteins of the present invention can be delivered in acarrier, as a solution, emulsion, colloid, or suspension, or as a drypowder, using any of a variety of devices and methods suitable foradministration by inhalation or other modes described here within orknown in the art.

Parenteral Formulations and Administration

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols, such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent, such as aqueous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthtetic mono- or di- or tri-glycerides. Parenteral administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446, entirely incorporated herein byreference.

Alternative Delivery

The invention further relates to the administration of at least oneIL-23 Ig derived protein or specified portion or variant by parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal,or transdermal means. An anti-IL-23 Ig derived protein or specifiedportion or variant compositions can be prepared for use for parenteral(subcutaneous, intramuscular, intravenous, intrarticular, etc.)administration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms, such as creams and suppositories; for buccal, orsublingual administration particularly in the form of tablets orcapsules; or intranasally particularly in the form of powders, nasaldrops or aerosols or certain agents; or transdermally particularly inthe form of a gel, ointment, lotion, suspension or patch delivery systemwith chemical enhancers, such as dimethyl sulfoxide, to either modifythe skin structure or to increase the drug concentration in thetransdermal patch (Junginger, et al. In “Drug Permeation Enhancement”;Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994,entirely incorporated herein by reference), or with oxidizing agentsthat enable the application of formulations containing proteins andpeptides onto the skin (WO 98/53847), or applications of electric fieldsto create transient transport pathways, such as electroporation, or toincrease the mobility of charged drugs through the skin such asiontophoresis, or application of ultrasound, such as sonophoresis (U.S.Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patentsbeing entirely incorporated herein by reference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably, at least one IL-23 Ig derivedprotein or specified portion or variant composition is delivered in aparticle size effective for reaching the lower airways of the lung orsinuses. According to the invention, at least one IL-23 Ig derivedprotein or specified portion or variant can be delivered by any of avariety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of Ig derived protein orspecified portion or variants are also known in the art. All suchdevices can use formulations suitable for the administration for thedispensing of Ig derived protein or specified portion or variant in anaerosol. Such aerosols can be comprised of either solutions (bothaqueous and non aqueous) or solid particles. Metered dose inhalers likethe Ventolin® metered dose inhaler, typically use a propellent gas andrequire actuation during inspiration (See, e.g., WO 94/16970, WO98/35888). Dry powder inhalers like Turbuhaler™ (Astra), Rotahaler®(Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed byInhale Therapeutics, and the Spinhaler® powder inhaler (Fisons), usebreath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like AERx™ Aradigm, the Ultravent® nebulizer(Mallinckrodt), and the Acorn II® nebulizer (Marquest Medical Products)(U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376), the above referencesentirely incorporated herein by reference, produce aerosols fromsolutions, while metered dose inhalers, dry powder inhalers, etc.generate small particle aerosols. These specific examples ofcommercially available inhalation devices are intended to berepresentative of specific devices suitable for the practice of thisinvention, and are not intended as limiting the scope of the invention.Preferably, a composition comprising at least one IL-23 Ig derivedprotein or specified portion or variant is delivered by a dry powderinhaler or a sprayer. There are several desirable features of aninhalation device for administering at least one Ig derived protein orspecified portion or variant of the present invention. For example,delivery by the inhalation device is advantageously reliable,reproducible, and accurate. The inhalation device can optionally deliversmall dry particles, e.g. less than about 10 μm, preferably about 1-5μm, for good respirability.

Administration of IL-23 Ig Derived Protein or Specified Portion orVariant Compositions as a Spray

A spray including IL-23 Ig derived protein or specified portion orvariant composition can be produced by forcing a suspension or solutionof at least one IL-23 Ig derived protein or specified portion or variantthrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, forexample, by an electric field in connection with a capillary or nozzlefeed. Advantageously, particles of at least one IL-23 Ig derived proteinor specified portion or variant composition protein delivered by asprayer have a particle size less than about 10 μm, preferably, in therange of about 1 μm to about 5 μm, and, most preferably, about 2 μm toabout 3 μm.

Formulations of at least one IL-23 Ig derived protein or specifiedportion or variant composition protein suitable for use with a sprayertypically include Ig derived protein or specified portion or variantcomposition protein in an aqueous solution at a concentration of about0.1 mg to about 100 mg of at least one IL-23 Ig derived protein orspecified portion or variant composition protein per ml of solution ormg/gm, or any range or value therein, e.g., but not lmited to, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm. The formulationcan include agents, such as an excipient, a buffer, an isotonicityagent, a preservative, a surfactant, and, preferably, zinc. Theformulation can also include an excipient or agent for stabilization ofthe Ig derived protein or specified portion or variant compositionprotein, such as a buffer, a reducing agent, a bulk protein, or acarbohydrate. Bulk proteins useful in formulating Ig derived protein orspecified portion or variant composition proteins include albumin,protamine, or the like. Typical carbohydrates useful in formulating Igderived protein or specified portion or variant composition proteinsinclude sucrose, mannitol, lactose, trehalose, glucose, or the like. TheIg derived protein or specified portion or variant composition proteinformulation can also include a surfactant, which can reduce or preventsurface-induced aggregation of the Ig derived protein or specifiedportion or variant composition protein caused by atomization of thesolution in forming an aerosol. Various conventional surfactants can beemployed, such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbitol fatty acid esters. Amounts will generally rangebetween 0.001 and 14% by weight of the formulation.

Especially preferred surfactants for purposes of this invention arepolyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, orthe like. Additional agents known in the art for formulation of aprotein, such as an IL-23 Ig derived proteins or specified portions orvariants, can also be included in the formulation.

Administration of IL-23 Ig Derived Protein or Specified Portion orVariant Compositions by a Nebulizer

Ig derived protein or specified portion or variant composition can beadministered by a nebulizer, such as jet nebulizer or an ultrasonicnebulizer. Typically, in a jet nebulizer, a compressed air source isused to create a high-velocity air jet through an orifice. As the gasexpands beyond the nozzle, a low-pressure region is created, which drawsa solution of Ig derived protein or specified portion or variantcomposition protein through a capillary tube connected to a liquidreservoir. The liquid stream from the capillary tube is sheared intounstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of Ig derived protein or specified portion or variantcomposition protein either directly or through a coupling fluid,creating an aerosol including the Ig derived protein or specifiedportion or variant composition protein. Advantageously, particles of Igderived protein or specified portion or variant composition proteindelivered by a nebulizer have a particle size less than about 10 μm,preferably, in the range of about 1 μm to about 5 μm, and, mostpreferably, about 2 μm to about 3 μm.

Formulations of at least one IL-23 Ig derived protein or specifiedportion or variant suitable for use with a nebulizer, either jet orultrasonic, typically include a concentration of about 0.1 mg to about100 mg of at least one IL-23 Ig derived protein or specified portion orvariant protein per ml of solution. The formulation can include agents,such as an excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one IL-23 Igderived protein or specified portion or variant composition protein,such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.Bulk proteins useful in formulating at least one IL-23 Ig derivedprotein or specified portion or variant composition proteins includealbumin, protamine, or the like. Typical carbohydrates useful informulating at least one IL-23 Ig derived protein or specified portionor variant include sucrose, mannitol, lactose, trehalose, glucose, orthe like. The at least one IL-23 Ig derived protein or specified portionor variant formulation can also include a surfactant, which can reduceor prevent surface-induced aggregation of the at least one IL-23 Igderived protein or specified portion or variant caused by atomization ofthe solution in forming an aerosol. Various conventional surfactants canbe employed, such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbital fatty acid esters. Amounts will generally rangebetween 0.001 and 4% by weight of the formulation. Especially preferredsurfactants for purposes of this invention are polyoxyethylene sorbitanmono-oleate, polysorbate 80, polysorbate 20, or the like. Additionalagents known in the art for formulation of a protein such as Ig derivedprotein or specified portion or variant protein can also be included inthe formulation.

Administration of IL-23 Ig Derived Protein or Specified Portion orVariant Compositions by a Metered Dose Inhaler

In a metered dose inhaler (MDI), a propellant, at least one IL-23 Igderived protein or specified portion or variant, and any excipients orother additives are contained in a canister as a mixture including aliquefied compressed gas. Actuation of the metering valve releases themixture as an aerosol, preferably, containing particles in the sizerange of less than about 10 μm, preferably, about 1 μm to about 5 μm,and, most preferably, about 2 μm to about 3 μm. The desired aerosolparticle size can be obtained by employing a formulation of Ig derivedprotein or specified portion or variant composition produced by variousmethods known to those of skill in the art, including jet-milling, spraydrying, critical point condensation, or the like. Preferred metered doseinhalers include those manufactured by 3M or Glaxo and employing ahydrofluorocarbon propellant.

Formulations of at least one IL-23 Ig derived protein or specifiedportion or variant for use with a metered-dose inhaler device willgenerally include a finely divided powder containing at least one IL-23Ig derived protein or specified portion or variant as a suspension in anon-aqueous medium, for example, suspended in a propellant with the aidof a surfactant. The propellant can be any conventional materialemployed for this purpose, such as chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the like.Preferably, the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one IL-23 Ig derived protein orspecified portion or variant as a suspension in the propellant, toprotect the active agent against chemical degradation, and the like.Suitable surfactants include sorbitan trioleate, soya lecithin, oleicacid, or the like. In some cases solution aerosols are preferred usingsolvents, such as ethanol. Additional agents known in the art forformulation of a protein can also be included in the formulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one IL-23 Ig derived protein or specified portion or variantcompositions via devices not described herein.

Oral Formulations and Administration

Formulations for oral administration rely on the co-administration ofadjuvants (e.g., resorcinols and nonionic surfactants, such aspolyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) toincrease artificially the permeability of the intestinal walls, as wellas the co-administration of enzymatic inhibitors (e.g., pancreatictrypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) toinhibit enzymatic degradation. The active constituent compound of thesolid-type dosage form for oral administration can be mixed with atleast one additive, including sucrose, lactose, cellulose, mannitol,trehalose, raffinose, maltitol, dextran, starches, agar, arginates,chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin,collagen, casein, albumin, synthetic or semisynthetic polymer, andglyceride. These dosage forms can also contain other type(s) ofadditives, e.g., inactive diluting agent, lubricant, such as magnesiumstearate, paraben, preserving agent, such as sorbic acid, ascorbic acid,.alpha.-tocopherol, antioxidant, such as cysteine, disintegrator,binder, thickener, buffering agent, sweetening agent, flavoring agent,perfuming agent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations may contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,871,753 are used to deliver biologically active agents orally and areknown in the art.

Mucosal Formulations and Administration

For absorption through mucosal surfaces, compositions and methods ofadministering at least one IL-23 Ig derived protein or specified portionor variant include an emulsion comprising a plurality of submicronparticles, a mucoadhesive macromolecule, a bioactive peptide, and anaqueous continuous phase, which promotes absorption through mucosalsurfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat.No. 5,514,670). Mucous surfaces suitable for application of theemulsions of the present invention can include corneal, conjunctival,buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal,and rectal routes of administration. Formulations for vaginal or rectaladministration, e.g. suppositories, can contain as excipients, forexample, polyalkyleneglycols, vaseline, cocoa butter, and the like.Formulations for intranasal administration can be solid and contain asexcipients, for example, lactose or can be aqueous or oily solutions ofnasal drops. For buccal administration, excipients include sugars,calcium stearate, magnesium stearate, pregelinatined starch, and thelike (U.S. Pat. No. 5,849,695).

Transdermal Formulations and Administration

For transdermal administration, the at least one IL-23 Ig derivedprotein or specified portion or variant is encapsulated in a deliverydevice, such as a liposome or polymeric nanoparticles, microparticle,microcapsule, or microspheres (referred to collectively asmicroparticles unless otherwise stated). A number of suitable devicesare known, including microparticles made of synthetic polymers, such aspolyhydroxy acids, such as polylactic acid, polyglycolic acid andcopolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers, such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof (U.S. Pat. Nos. 5,814,599).

Prolonged Administration and Formulations

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid, such as phosphoric acid, sulfuric acid, citric acid, tartaricacid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and thelike; (b) a salt with a polyvalent metal cation, such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmiumand the like, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g., a zinc tannate salt. Additionally, the compounds ofthe present invention or, preferably, a relatively insoluble salt, suchas those just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulationin a slow degrading, non-toxic, non-antigenic polymer, such as apolylactic acid/polyglycolic acid polymer, for example, as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts, such as those described above, can also be formulatedin cholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g., gas orliquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES OF THE INVENTION Example 1 Generation, Cloning and Expressionof an Anti-IL-23 Immunoglobulin Derived Protein in Mammalian Cells

Anti-IL-23 Ig derived proteins are generated using known methods, suchas murine or transgenic mice expressing human antibodies that areimmunized with human IL-23, and for which B cells are isolated, clonedand selected for specificity and inhibiting activity for IL-23(preferably with little or no inhibition of IL-12 activity) using knownmethods and assays, e.g., as known in the art and as described herein(see, e.g., www.copewithcytokines.de, under IL-23 and IL-12, fordescription and references to IL-23 proteins, IL-23 assays and IL-12assays, entirely incorporated herein by reference, as known in the art).Alternatively, portions of the IL-12 beta 1 receptor are cloned andfused with antibody fragments to generate receptor fusion proteins thatblock binding of IL-23 to its receptors but which do not inhibit bindingof IL-12 to its receptors, as known in the art.

Clones expressing IL-23 specific antibodies or fusion proteins, such asthe anti-IL-23 Ig derived proteins of the present invention are selectedso that they neutralize or inhibit at least one IL-23 activity and whichdo not substantially inhibit at least one IL-12 activity.

The heavy chain, light chain CDRs, variable regions, or variable andconstant regions are cloned and put into appropriate expression vectors.A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, the Igderived protein or specified portion or variant coding sequence, andsignals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription canbe achieved with the early and late promoters from SV40, the longterminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI andthe early promoter of the cytomegalovirus (CMV). However, cellularelements can also be used (e.g., the human actin promoter). Suitableexpression vectors for use in practicing the present invention include,for example, vectors, such as pIRES1neo, pRetro-Off, pRetro-On, PLXSN,or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1 (+/−), pcDNA/Zeo(+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL and PMSG (Pharmacia,Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) andpBC12MI (ATCC 67109). Mammalian host cells that could be used includehuman Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1,Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamsterovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded Ig derived protein or specified portion or variant. The DHFR(dihydrofolate reductase) marker is useful to develop cell lines thatcarry several hundred or even several thousand copies of the gene ofinterest.

Another useful selection marker is the enzyme glutamine synthase (GS)(Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al.,Bio/Technology 10: 169-175 (1992)). Using these markers, the mammaliancells are grown in selective medium and the cells with the highestresistance are selected. These cell lines contain the amplified gene(s)integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cellsare often used for the production of Ig derived protein or specifiedportion or variants.

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of IL-23 Ig derived protein orspecified portion or variant. Plasmid pC4 is a derivative of the plasmidpSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the mouseDHFR gene under control of the SV40 early promoter. Chinese hamsterovary—or other cells lacking dihydrofolate activity that are transfectedwith these plasmids can be selected by growing the cells in a selectivemedium (e.g., alpha minus MEM, Life Technologies, Gaithersburg, Md.)supplemented with the chemotherapeutic agent methotrexate. Theamplification of the DHFR genes in cells resistant to methotrexate (MTX)has been well documented (see, e.g., F. W. Alt, et al., J. Biol. Chem.253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys. Acta1097:107-143 (1990); and M. J. Page and M. A. Sydenham, Biotechnology9:64-68 (1991)). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene, it is usually co-amplified and over-expressed. It isknown in the art that this approach can be used to develop cell linescarrying more than 1,000 copies of the amplified gene(s). Subsequently,when the methotrexate is withdrawn, cell lines are obtained that containthe amplified gene integrated into one or more chromosome(s) of the hostcell.

The plasmid pC4 (and also pC1) contains for expressing the gene ofinterest the strong promoter of the long terminal repeat (LTR) of theRous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985))plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530(1985)). Downstream of the promoter are BamHI, XbaI, and Asp718restriction enzyme cleavage sites that allow integration of the genes;the multiple cloning sites facilitate cloning of the gene of interest.Behind these cloning sites, the plasmid contains the 3′ intron andpolyadenylation site and termination signal of the rat preproinsulingene. Other high efficiency promoters can also be used for theexpression, e.g., the human b-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems andsimilar systems can be used to express the IL-23 in a regulated way inmammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA89: 5547-5551 (1992)). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes, can beused as well. Stable cell lines carrying a gene of interest integratedinto the chromosomes can also be selected upon co-transfection with aselectable marker, such as gpt, G418 or hygromycin. It is advantageousto use more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete IL-23 Ig derived protein orspecified portion or variant is used, corresponding to HC and LCvariable regions of an IL-23 Ig derived protein of the presentinvention, according to known method steps. Isolated nucleic acidencoding a suitable human constant region (i.e., HC and LC regions) isalso used in this construct (e.g., as provided in vector p1351).

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmidpSV2neo contains a dominant selectable marker, the neo gene from Tn5encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about 10-14 days, single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM.Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

The completely human anti-IL-23 protein Ig derived proteins are furthercharacterized. Several of the generated Ig derived proteins are expectedto have affinity constants between 1×10⁹ and 9×10¹². Such highaffinities of these fully human monoclonal Ig derived proteins make themsuitable for therapeutic applications in IL-23 protein-dependentdiseases, pathologies or related conditions.

Example 2 Comparison of the Therapeutic Efficacy of Anti-IL-12P35 andAnti-IL-12/23P40 Antibodies in Murine Experimental AutoimmuneEncephalomyelitis (EAE)

Summary: This set of studies was performed to investigate thetherapeutic efficacy of IL-12 or IL-12/IL-23 specific neutralization ina mouse model for multiple sclerosis, experimental autoimmuneencephalomyelitis (EAE). Neutralizing rat anti-mouse monoclonalantibodies (mAbs) specific for the p35 subunit of IL-12 or the p40subunit that is shared between IL-12 and IL-23 were administered eitherprior to disease induction, prior to disease onset, or after disease wasongoing. In all cases, only anti-p40 demonstrated therapeutic potential.These data suggest that IL-23 is the predominant contributor to diseasepathogenesis in this autoimmune model.

Abbreviations: IL Interleukin mAb Monoclonal antibody EAE Experimentalautoimmune encephalomyelitis Th T helper cell IFNγ Interferon gamma csClinical score MBP Myelin basic protein PK Pharmacokinetics

Introduction: Biologically active IL-12 exists as a heterodimercomprised of 2 covalently linked subunits of 35 (p35) and 40 (p40) kiloDaltons. Several lines of evidence have demonstrated that IL-12 caninduce robust Th1 immune responses that are characterized by productionof IFNγ and IL-2 from CD4⁺ T cells. Inappropriate Th1 responses, andthus IL-12 expression, are believed to correlate with manyimmune-mediated diseases, such as multiple sclerosis, rheumatoidarthritis, inflammatory bowel disease, insulin-dependent diabetesmellitus, and uveitis. In animal models, IL-12 neutralization was shownto ameliorate immune-mediated disease. However, these studiesneutralized IL-12 through its p40 subunit. The recent description ofIL-23 (1), a heterodimeric cytokine that shares the p40 subunit, made itimportant to determine whether previous findings were due to IL-12 orIL-23 activity. Therefore, p35 and p40 specific neutralization werecompared in a mouse model of autoimmunity, experimental autoimmuneencephalomyelitis (EAE). Neutralizing antibodies specific for IL-12p35had no effect on EAE progression. In contrast, neutralization of bothIL-12 and IL-23 with an anti-p40 mAb suppressed clinical signs of EAE,whether antibody was administered before or after Th1 differentiation.This data suggests that the activity of anti-p40 treatment in EAE isbased solely on neutralization of IL-23.

Methods and Materials:

Mice:

Female C3H/HEB/FEJ mice (Jackson Laboratories, Bar Harbor, Me.) wereused in pharmacokinetic analyses. For EAE studies, female B10.PL(H-2^(u)) mice were purchased from the Jackson Laboratories, and wereused between 6-8 weeks of age. All animals were maintained according toIACUC guidelines under approved protocols.

Antibodies:

C17.8 (rat anti-mouse IL-12/IL-23p40, IgG2a), and C18.2 (rat anti mouseIL-12p35, IgG2a) hybridomas provided by Dr. Giorgio Trinchieri and theWistar Institute (Philadelphia, Pa.). Ascites was generated at HarlanBioproducts (Indianapolis, Ind.) and purified by protein G affinity.

Serum PK of Rat Anti-Mouse Antibodies:

Female C3H/HEB/FEJ mice, approximately 20-25 grams, were individuallyweighed and treated with a single 5 mg/kg intraperitoneal dose of ¹²⁵Ilabeled antibody (C17.8, C18.2), with a constant dose volume/mouse of 10mL/kg. Retro-orbital bleeds were taken from anesthetized mice at 30minutes, 6 and 24 hours, 4, 7, 11 and 18 days. Blood samples wereallowed to stand at room temperature for at least 30 minutes, but nolonger than 1 hour, and were then centrifuged at approximately2,500-3,500 rpm for 10-15 minutes. Approximately 50 uL aliquots of eachserum sample were counted for 125Iusing a LKB Compugamma 1282 counter(Wallac, Gaithersburg, Md.). 10 mL aliquots of the injectates were alsocounted. The average fraction of injected counts at each time point wascalculated and multiplied by the total mg of antibody injected todetermine the total mg remaining in the serum at each time point. Datais shown as the mean mg of mAb in the sera +/−s.d. with 5-10 animals ineach group.

EAE Induction and Scoring:

For EAE induction, female B10.PL mice were injected subcutaneously overfour sites on the back with a total of 100 μl of CFA (containing 200 μgMycobacterium tuberculosis Jamaica strain) combined with 200 μg guineapig-MBP (Sigma). Mice also received 200 ng pertussis toxin (ListBiological, Campbell, Calif.) i.p. in 0.2 ml PBS at the time ofimmunization and 48 hours later. Mice received i.p. injections of C17.8(anti-IL-12p40) or C18.2 (anti-IL-12p35) monoclonal antibodies dilutedto 100 mg/kg (C18.2) or 20 mg/kg (C17.8) in PBS, on indicated days.Control mice received PBS or Rat IgG (Biosource) at 20 mg/kg in PBS.

Animals that demonstrated clinical signs (cs) were scored as follows:limp tail or waddling gait with tail tonicity 1, waddling gait with limptail (ataxia) 2, ataxia with partial limb paralysis 2.5, full paralysisof one limb 3, full paralysis of one limb with partial paralysis ofsecond limb 3.5, full paralysis of two limbs 4, moribund 4.5, death 5.Animals that scored a 5 were not included in the mean daily cs analysisfor the rest of the experiment. Daily cs are averaged for the group, andmean incidence, day of onset, highest acute cs, cumulative cs, cs/day,number of relapses and relapse severity ±sem are described. Meancumulative cs per group was calculated by averaging the sum of dailyclinical scores for individual animals. cs/day was calculated bydividing the cumulative cs by the number of days the animal remained inthe study. To determine the mean day of onset, animals not developingEAE were not included in the analysis. To determine the mean highest cs,mice not developing EAE were assigned a value of “0” and included in theanalysis. Relapses were defined by a full point drop in clinical scoresustained for at least 2 observed days followed by a full point increasein clinical score sustained for at least 2 observed days.

Results and Discussion: Anti-p35 and Anti-p40 Antibodies have IdenticalPharmacokinetics

To establish the clearance rates of anti-p40 and anti-p35 antibodies,normal mice were injected with a single 5 mg/kg dose of ¹²⁵I labeledantibodies and circulating levels were measured for 11 days postantibody administration. Anti-p35 and anti-p40 had overlappingpharmacokinetics, demonstrating that clearance rates are identical innormal mice (2). The expected clearance rate of each mAb isapproximately 7-10 days. Although this is a single dose PK study, thesedata support once weekly dosing for in vivo studies.

Only Anti-p40 Treatment Prior to EAE Induction is Protective

To determine the relative roles of IL-12 and IL-23 in immune-mediateddiseases, we utilized a murine model for multiple sclerosis, relapsingexperimental autoimmune encephalomyelitis (EAE). Upon EAE induction withmyelin basic protein (MBP) in adjuvant, B10.PL mice typically exhibit aninitial episode of paralysis (acute disease), then recover eitherpartially or completely and progress through multiple relapses and/orchronic EAE. It has long been assumed that EAE is dependent upon IL-12expression since IL-12 is believed to be a primary mediator of Th0 toTh1 differentiation. However, to distinguish the potential role of IL-23in EAE induction, neutralizing concentrations of anti-p40 (IL-12 andIL-23) or anti-p35 (IL-12 only) antibodies were established one dayprior to immunization for EAE (Day-1). Onset of disease can vary betweenanimals; therefore, treatment was repeated 7 and 14 days later to ensurethat anti-p35 and IL-p40 antibodies were present during Th1differentiation. Several in vitro neutralization studies havedemonstrated that the anti-40 mAb is 5 times more effective inneutralizing IL-12 than the anti-p35 mAb (data not shown). Therefore,the dose of anti-p35 mAb was adjusted to be 5 fold higher than anti-p40in all EAE experiments. In two separate experiments, mice treated withRat IgG isotype control antibody (20 mg/kg) or anti-p35 (100 mg/kg) didnot demonstrate protection from disease. It is important to note thatperipheral administration of a non-specific control antibody (Rat IgG)did not alter the clinical course of disease when compared tonon-treated mice with EAE. In both studies, mice treated with anti-p40mAb (20 mg/kg) exhibited nearly complete inhibition of EAE clinicalsigns. Remarkably, suppression of disease extended beyond the expectedrate of antibody clearance through 70 days post EAE induction. In eachexperiment, only one animal treated with anti-p40 exhibited twoconsecutive days of EAE clinical signs, and each demonstrated a lateonset and significantly lower acute clinical scores, cumulative clinicalscores, and no relapses in disease (Table 1). These results demonstratethat neutralization of IL-12 and IL-23 through the shared p40 subunitprovided nearly complete protection from EAE. In contrast, specificneutralization of IL-12 only via anti-p35 was ineffective. These datastrongly suggest that EAE is not mediated by IL-12.

Only Anti-p40 Treatment Just Prior to Disease Onset is Protective

Although prophylactic treatment completely protected mice from EAE, itremained to be determined if IL-12 specific neutralization would beprotective once the Th1 population was established in vivo. Therefore,in a separate set of experiments, mice were treated with either acontrol antibody (Rat IgG), anti-p35, or anti-p40 monoclonal antibodiesten days after EAE induction, but prior to disease onset. Since typicalimmune responses occur within 7 days, this time point should reflect theeffects of anti-IL-12 or anti-IL-23 mAbs on differentiated Th1 cells.EAE onset can vary between animals, therefore treatment was repeated 7and 14 days later to ensure that anti-p35 and anti-p40 antibodies werepresent during the onset of disease. In two separate experiments, micetreated with isotype control antibody (20 mg/kg) or anti-p35 (100 mg/kg)were not protected from disease, when compared to untreated EAE mice.However, mice treated with anti-p40 mAb (20 mg/kg) were significantlyprotected from EAE. As shown in the previously described studies,disease suppression was observed well beyond the time required forclearance of peripherally administered antibody through day 70 post EAEinduction. Considering that antibody was not administered until afterTh1 differentiation (day 10), it was not surprising that diseaseincidence, day of onset, and the highest clinical score during acute EAEwere not different in any group (Table 2). However, in both experiments,mice receiving anti-p40 exhibited significantly lower cumulativeclinical scores, clinical scores per day, and relapse severity.

Only Anti-p40 Treatment During Established EAE is Protective

The most difficult, but clinically relevant, hurdle for any therapy isto suppress established disease. Therefore another set of experimentswas performed in which mice were immunized for EAE, then divided intotreatment groups once disease was ongoing. Approximately 30 days postEAE induction, mice had progressed through the acute phase of disease.At this time, animals were divided into groups with comparablecumulative and daily clinical scores. Treatment was repeated 7 and 14days later to ensure that antibodies were available in neutralizingconcentrations during the transition from acute to chronic orremitting-relapsing disease. Only anti-p40 treatment (20 mg/kg)ameliorated disease when compared to either isotype control antibody (20mg/kg) or anti-p35 (100 mg/kg) treated animals. Disease suppression wasobserved through day 80 post EAE induction. In both experiments,analysis from the first day of treatment through day 80 demonstratedthat mice receiving anti-p40 exhibited lower cumulative clinical scores,clinical scores per day, and the least highest clinical score posttreatment. These data suggest that not only is IL-23 likely to mediateTh1 differentiation (Table 1) and EAE induction (Table 2), but IL-23also contributes to the effector phase of chronic immune-mediated (e.g.,autoimmune) responses (Table 3). Therefore, anti-p40 treatment can offertherapy at any time in the progression of immune-mediated diseases.

Conclusions

The understanding of the role of IL-12 in immune function has been basedon studies of the p40 subunit of IL-12. Therefore, a side-by-sidecomparison of neutralization of the IL-12 specific p35 subunit versusthe p40 subunit shared between IL-12 and IL-23 was conducted in ananimal model of autoimmune disease. Neutralization via anti-p40significantly inhibited EAE when mAb was administered at any time point.However, IL-12 specific neutralization was completely ineffective.Therefore, our data shows that IL-12 does not contribute to thisautoimmune model and that IL-23 is expected be the more prominentmediator of autoimmune T cell responses.

Example 3 IL-23 Mediates Experimental Autoimmune Encephalomyelitis

Materials and Methods

Animals:

Female B10.PL mice (Jackson Laboratories, Bar Harbor, Me.) and femaleC57BL/6 mice (Charles River Laboratories, Raleigh N.C.) between 6-8weeks of age were used and maintained according to IACUC guidelinesunder approved protocols.

Antibodies

Rat monoclonal antibodies to mouse IL-23 were developed at Centocor(Malvern, Pa.). Negative rat IgG (from Biosource, Camarillo, Calif.) wasused as a control. Neutralizing rat anti-mouse p40 (C17.8), and ratanti-mouse IL-12 (C18.2) antibodies were provided by Dr. GiorgioTrinchieri and the Wistar Institute (Philadelphia, Pa.). Ascites wasgenerated at Harlan Bioproducts (Indianapolis, Ind.) and antibodies werepurified by protein G affinity chromatography.

Cytokines

Recombinant murine IL-12 was obtained from R&D Systems (Minneapolis,Minn.). Recombinant hIFN-γ and human IL-2 were obtained from Peprotech(Rocky Hill, N.J.). Murine IL-23 was generated using transienttransfection technology and Immobilized Metal Affinity Chromatography(IMAC). Briefly, separate expression constructs for murine p40 andmurine p 19-His were co-transfected into HEK 293E cells usingLipofectamine 2000 (Invitrogen, Carlsbad, Calif.) as suggested by themanufacturers instructions. Alternatively, a linked IL-23 construct wasgenerated as described and transfection of HEK 293E cells was performed.Twenty-fours hours post-transfection, the growth medium was replacedwith serum-free 293 SFMII (Invitrogen) and left to condition for 5 days.The media was then removed, centrifuged, and processed by IMAC usingTALON resin (BD Biosciences, Palo Alto, Calif.). His-tagged proteinswere eluted with 150 mM EDTA, then dialyzed against PBS, concentrated,filtered, and stored at −80° C. Bioactivity of both co-transfected andlinked IL-23 was verified by splenocyte IL-17 protein production asdescribed below.

IL-12 and IL-23 ELISA

Murine IL-12 and murine IL-23 (1 μg/ml) were coated overnight on NuncMaxisorp plates in PBS. After the plates were washed and blocked, ratanti-mouse p40, rat anti-mouse IL-12, and rat anti-mouse IL-23antibodies were titrated and allowed to bind for 2 hours. Bound proteinwas detected using 1:10,000 HRP-conjugated goat anti-rat IgG antibody(from Jackson Immuno Research, West Grove, Pa.) followed by substrate.Data is shown as the mean optical density of replicate wells.

IL-12 Neutralization

Non-adherent human peripheral blood mononuclear cells (PBMC) werecultured for four days with 5 μg/ml PHA (Lectin, Phaseolus vulgaris,Sigma, St. Louis, Mo.) in complete RPMI-1640 (Invitrogen) with 10%heat-inactivated fetal bovine serum (JRH, Lenexa, Kans.), 1% L-glutamine(JRH), 100 Units/ml penicillin and 100 μg/ml streptomycin (Invitrogen).Cells were harvested, washed, then cultured with rhIL-2 (10 units/ml) inthe presence of murine IL-12 (1 ng/ml) either alone or pre-incubatedwith tested antibodies for 22 hours. Supernatants were analyzed forhuman IFNγ protein levels by luminescence immunoassay using anti-IFNγantibodies generated at Centocor.

IL-23 Neutralization

Single cell suspensions were prepared from spleens of C57BL/6 mice.2×10⁶ cells/ml were cultured in complete RPMI with 10 U/ml rhIL-2(Peprotech) and 1 ng/ml mouse IL-23, either alone or pre-incubated withtested antibodies for 3 days. Supernatants were collected and analyzedfor IL-17 protein by ELISA (R&D Systems) per the manufacturer'sinstructions.

EAE Analysis

Female B10.PL mice were injected s.c. over four sites on the back with atotal of 100 μl of complete Freunds adjuvant (CFA) combined with 200 μgguinea pig-myelin basic protein (MBP) (Sigma). Mice also received 200 μgpertussis toxin (List Biological, Campbell, Calif.) i.p. in 0.2 ml PBSat the time of immunization and 48 hours later. Mice received i.p.injections of anti-p40, anti-IL-12, or anti-IL-23 monoclonal antibodiesdiluted to 100 mg/kg (anti-IL-12), 20 mg/kg (anti-p40, anti-IL-23), or50 mg/kg (anti-IL-23) in PBS, on indicated days. Control mice wereeither not treated or received Rat IgG (Biosource, Camarillo, Calif.) at20 mg/kg in PBS.

Animals that demonstrated clinical signs (cs) were scored as follows:limp tail or waddling gait with tail tonicity 1, waddling gait with limptail (ataxia) 2, ataxia with partial limb paralysis 2.5, full paralysisof one limb 3, full paralysis of one limb with partial paralysis ofsecond limb 3.5, full paralysis of two limbs 4, moribund 4.5, death 5.Scores for animals that were sacrificed or scored a 5 were not includedin the mean daily cs analysis for the rest of the experiment. Daily csare averaged for the group, and incidence, mortality, day of onset,highest acute cs, cumulative cs, cs/day, number of relapses and relapseseverity ±sem are described. Mean cumulative cs per group was calculatedby averaging the sum of daily clinical scores for individual animals.Cs/day was calculated by dividing the cumulative cs by the number ofdays the animal remained in the study. To determine the mean day ofonset, animals not developing EAE were not included in the analysis. Todetermine the mean highest acute cs, mice that never developed EAE wereassigned a value of “0” and included in the group mean. Relapses weredefined by a full point drop in clinical score sustained for at least 2observed days followed by a full point increase in clinical scoresustained for at least 2 observed days. To determine the mean number ofrelapses per group, mice not demonstrating a defined relapse wereassigned a value of “0” and included in the group mean. To determine themean relapse severity, the highest clinical score of each relapse eventwas averaged and animals that did not relapse were not included in theanalysis.

For ex vivo EAE analysis, spleens and peripheral lymph nodes (inguinal,axillary, brachial, and cervical) were harvested from each animal ondays 10, 17, 24, or 32 post EAE induction. Single cell suspensions(5×10⁵/well) were prepared from individual animals, washed twice, thencultured in vitro in RPMI complete for 72 hours with 40 μg/ml MBP, 5μg/ml ConA, or media alone and proliferation was measured using ATPLite(Perkin Elmer, Boston, Mass.). Data is represented as a stimulationindex, which is the mean proliferation to MBP divided by the meanproliferation to media alone. Splenocytes and lymph node cells were alsocultured at 4×10⁶ cells/ml with 40 μg/ml MBP or media alone for 48 hoursand supernatants were tested for IFNγ, IL-17, IL-4, IL-5, and IL-10proteins by ELISA, according to the manufacturer instructions (R&DSystems). Even though minimal cytokine levels were detected inmedia-only cultures, those values were subtracted from the levels foundin MBP-stimulated cultures so that the data presented represents onlyantigen-specific cytokine production.

For histopathologic examination and ranking, mouse brains and spinalcolumns were fixed in 10% buffered formalin by emersion. After fixation,the brains were sliced coronally into 4 segments. Spinal columns weredecalcified in 5% EDTA and then sliced sagitally into 5 segments. Thetissues were processed and embedded in paraffin using routine methods.Tissue blocks were sectioned at 5 μm, and stained with hematoxylin andeosin (H&E) or Luxol Blue-Cresyl Echt Violet (Poly Scientific, BayShore, N.J.). Additional sections were stained immunohistochemically forglial fibrillary acidic protein (GFAP) (BioGenex, San Ramon, Calif.).Sections were blinded and ranked based on the extent of inflammation.Brains and spinal cords were analyzed separately.

Results

IL-23 specific neutralization ameliorates EAE

To confirm that neutralization of only IL-23 will provide effectivetherapy for EAE, monoclonal antibodies to mouse IL-23 were generated. Asshown in FIG. 1A, an antibody specific for mouse IL-23 that demonstratedno reactivity with mouse IL-12 was identified. Subsequent studies haveshown that the anti-IL-12 and anti-IL-23 antibodies do not cross reacteven when 100 ng/ml of the opposite cytokine is present. As shown inFIG. 1B, the anti-IL-23 specific antibody binds to the p40 subunit ofIL-23 and does not bind to the p19 subunit. Accordingly, it is IL-23p40specific.

Since it was recently shown that IL-23 will induce IL-17 production,these antibodies were tested for their ability to neutralize IL-23bioactivity. As shown in FIG. 1C, the IL-23 specific antibody inhibitsIL-17 production with similar potency as anti-p40. In contrast, theanti-IL-12 antibody demonstrated no effect on IL-17 levels. Lastly, toconfirm that the anti-IL-23 antibody does not interfere with IL-12function, the antibodies' ability to inhibit IFNγ production in T cellcultures was tested. As previously demonstrated, anti-IL-12 and anti-p40inhibited IFNγ production, however, the anti-IL-23 antibody had noeffect on IFNγ levels (FIG. 1D). Therefore, a neutralizing anti-mouseIL-23 antibody that does not bind IL-12 or inhibit IL-12 mediatedresponses has been developed.

The anti-IL-23 and anti-p40 antibodies were compared for in vivoinhibition of EAE. Mice were dosed with a control antibody, anti-p40, oranti-IL-23 either ten days after EAE induction or during establishedEAE. In both paradigms, mice treated with anti-IL-23 demonstratedclinical suppression of EAE comparable to that of anti-p40 treatedanimals (FIG. 6A). Mice receiving anti-p40 or anti-IL-23 exhibited alater day of onset, reduced severity of acute disease and subsequentrelapses, and lower clinical scores per day. These results demonstratethat even in mice that have not been genetically manipulated, IL-23, andnot IL-12, is responsible for encephalitogenic T cell responses in EAE.

IL-23 Neutralization Prevents EAE Pathology in the CNS

EAE presents as an ascending hind limb paralysis and is therefore scoredfor severity by deficits in motor function. However, the cause of thisimpairment can only be observed by assessing pathology within the brainand spinal cord. Therefore, a separate study was performed in which micewere immunized for EAE, then treated with control Rat IgG, anti-IL-12,anti-p40, or anti-IL-23 antibodies on days 10 and 17. The mice werescored daily for EAE clinical signs and then sacrificed on day 24 bycardiac perfusion. Sections of the brain and spinal cord were blindedand ranked from least to most severe, then correlated to the clinicalscore of the animal on the day of sacrifice.

The severity of spinal cord pathology correlated with the clinical scoreseverity, whereas brain pathology did not. This is not surprising sinceclinical scoring is defined by motor ability, which is primarily ameasurement of spinal cord function. Histopathology rankings were thensub-divided into treatment groups to assess differences after 2 in vivoantibody treatments (day 24). All treatment groups, includinganti-IL-12, had lower pathology rankings than the Rat IgG treatedcontrol animals. However, it is important to note that with treatmentparadigms that are initiated 10 days post EAE induction, clinicalprotection with anti-p40 or anti-IL-23 is not typically observed untilday 30 or later (FIG. 1D).

There were remarkable differences in spinal cord inflammation,demyelination, and astrocyte gliosis when the Rat IgG control andanti-IL-23 groups were compared. Serial sections of thoracic spinal cordfrom mice representative of the median clinical score ranking of thecontrol (rat IgG) group and anti-IL-23 treated group were stained: (1)by H&E to evaluate cellular infiltration (FIGS. 2A and 2B at 40 timesmagnification) in which FIG. 2A represents the control group and FIG. 2Brepresents the anti-IL-23 treated group; (2) by Luxol Fast Blue toevaluate demyelination (FIGS. 2C and 2D at 200 times magnification) inwhich FIG. 2C represents the control group and FIG. 2D represents theanti-IL-23 treated group; and (3) by GFAP to evaluate astrocyte gliosis(FIGS. 2E and 2F at 200 times magnification) in which FIG. 2E representsthe control group and FIG. 2F represents the anti-IL-23 treated group.These data confirm that the clinical protection that is observed afteranti-IL-23 therapy is a result of partial protection from CNS pathology.

IL-23 Specific Neutralization Leaves IL-12 Pathways Intact

Mouse strains that are susceptible to Leishmania major are unable tocontain the organism, whereas resistant strains will localize theorganism and lesions will spontaneously heal. It has been previouslydemonstrated that administration of anti-p40 antibodies decreases theability of resistant mice to clear Leishmania major, presumably due to adiminished Th1 response. To determine the specific role of IL-12,anti-IL-12 and anti-p40 antibodies in the L. major model in a resistantmouse strain were compared. Remarkably, despite the 5-fold lowerneutralizing capacity of anti-IL-12 antibody, each antibody equallydelayed immunity to L. major for 50 days (FIG. 6B). IL-12 specific invivo neutralization seemed to have a longer lasting effect in delayingL. major immunity, when compared to anti-p40 treatment even thoughlesions in animals from both treatment groups were nearly resolved bythe end of the observation period (day 85).

Utilizing the neutralizing murine IL-23 antibody, the in vivo responsesto L. major upon IL-23 neutralization were investigated. As shown inFIG. 6C, anti-IL-23 did not alter immunity to L. major when compared toRat IgG treated animals. In contrast, IL-12 neutralization via IL-12 orp40 specific antibodies did inhibit L. major immunity, as observedpreviously (FIG. 6B) in a slightly different model of L. majorinfection. These results confirm that neutralization of IL-23 canprovide therapy for immune-mediated disease, while leaving IL-12pathways of pathogen immunity intact.

Example 4 IL-23p40 Epitope

IL-23 functions by binding to a 2-chain receptor complex that isexpressed on the surface of T cells, natural killer (NK) cells, andpotentially antigen presenting cells. The IL-12Rbeta(β)-1 chain is anon-signaling receptor chain that is responsible for IL-12/23p40binding. Therefore, it is logical that IL-12Rβ1 is a component of boththe IL-12 and the IL-23 receptor complexes. However, each receptorcomplex has a signaling subunit that is specific for each respectivecytokine.

IL-12Rβ1 binds to the p35 subunit of IL-12 and is responsible forinitiating IL-12 specific cellular responses. In contrast, IL-23R bindsto IL-23p19 and is responsible for IL-23 specific cellular responses. Itis understood that neutralizing antibodies that bind to the p40 subunitof IL-12 or IL-23, or the shared IL-12RP 1, can inhibit the bioactivityof both cytokines by preventing cytokine/receptor interaction. However,it was assumed that specific inhibition of IL-12 could only be achievedthrough antibodies to IL-12p35 or IL-12Rβ2 (see FIG. 3 showing theunderstood mechanisms for specific antibody neutralization of IL-12and/or IL-23). Likewise, it was assumed that specific inhibition ofIL-23 could only be achieved through antibodies to IL-23p 19 or IL-23R(FIG. 3).

The present invention provides novel p40 epitopes that are available inan IL-23 (p19/p40) complex and not in IL-12 (p35/p40) and an antibodywith no reactivity to IL-12 that can bind to p40 epitope(s) andspecifically inhibit IL-23 through interruption of IL-23 p40 binding toIL-12Rβ1. In addition, the present invention provides methods ofgenerating anti-human or murine IL-23p40 binding agents. These agentswould provide IL-23 specific therapy for immune-mediated disease withoutinterrupting IL-12 mediated pathways.

Experimental Results:

Rats were immunized with DNA encoding murine IL-23p 19, thensubsequently boosted with murine IL-23 protein. Hybridomas weregenerated and supernatants were tested for IL-23 and IL-12 reactivity.Hybridomas that produced IL-23 positive and IL-12 negative antibodieswere subcloned twice to generate monoclonal cultures. Four ratanti-mouse monoclonal antibodies (mAb) were identified that bound IL-23and not IL-12. These mAbs were further characterized for neutralizationpotency. One mAb was identified that had no reactivity to murine IL-12,yet potently neutralized the bioactivity of murine IL-23.

The mAb was further analyzed for IL-23 subunit specificity, and it wasdiscovered to bind to the p40 monomer. Therefore, through monoclonalantibody technology, a functional p40 epitope that is present in anIL-23 (p19/p40) complex that is not present in an IL-12 (p35/p40)complex has been identified. While it might not be unexpected that 2heterodimers would present unique epitopes, it is unexpected that theunique epitopes would be critical for interaction with the IL-12Rβ1receptor chain shared by IL-12 p40 and IL-23 p40. In contrast, it wouldbe assumed that critical IL-12 μl I-binding epitopes would be conservedbetween IL-12 and IL-23.

Accordingly, the present invention identifies IL-12Rβ1-binding epitopesthat are unique to IL-23 and a novel agent and mechanism for IL-23specific neutralization that is unexpected from the present state of theart. FIG. 4 illustrates an example of an IL-12Rβ1 binding p40 epitopethat would only be available on IL-23.

The anti-murine IL-23 p40 antibody does not bind to human IL-12, humanIL-23, or human p40. Therefore, potential p40 epitopes were identifiedby comparing sequence and structural information for murine IL-12 versusmurine IL-23, then murine IL-23 versus human IL-23.

The crystal structure of the human IL-12 has been reported (shown inFIG. 5). The p35 subunit is a four-helix bundle protein that iscovalently linked to the p40 subunit through the inter-chain disulfidebond. It has been predicted that p19 subunit is most likely also afour-helix bundle protein. Based on sequence homology and the pattern ofsecondary structures of p 19 and p35, p19 should bind p40 in a similarmanner. In all likelihood, the bottom portion of the structurerepresents both p35 (observed) and p19 (predicted). Because of thesignificant size differences between p35 and p19, the surface(s) on p40that is available for antibody binding will be different between IL-12and IL-23.

Based upon the binding data, it was hypothesized that the antibody'sbinding epitope must be (a) exposed on p40 surface, (b) near or coveredby the p35/p19 subunits in the structures, (c) not close to the p35/p19helices since they are most likely very similar between p 19 and p35 andwould prevent antibody access, and/or (d) different between the humanand murine in amino acid sequences in these regions. Two such regionsare circled in FIG. 4 which shows the predicted human p40 epitopes forIL-23 specific binding. Three sequence segments within the 2 identifiedregions contain differences between human and murine sequences. Twosequences are located in domain 2 (D2) (amino acids 84-212 of SEQ IDNO:1) and one is located in domain 3 (D3) (amino acids 213-306 of SEQ IDNO:1) of p40; domain 1 (D1) comprises amino acids 1-83 of SEQ ID NO:1.The three potential epitope segments are underlined and labeled as Seg1,Seg2 and Seg3 in the sequence alignment between the human and murine p40(Table 5 below). Any agent that binds these epitopes on the human ormurine IL-23p40 proteins could offer specific inhibition of IL-23bioactivity, without binding IL-12 and inhibiting IL-12.

TABLE 5 Predicted sequences for unique IL-23 p40 epitopes IL-12/23 p40hu_p40 mu_p40 (1) (1)

hu_p40 mu_p40 (61) (61)

hu_p40 mu_p40 (121) (118)

hu_p40 mu_p40 (180) (178)

hu_p40 mu_p40 (240) (237)

hu_p40 mu_p40 (292) (296)

This epitope information can be used to generate and/or select IL-23p40specific binding agents. Epitope-directed mutant p40 proteins can begenerated by exchanging murine and human seg1, seg2, and/or seg3sequences into native murine or human p40 proteins. Alternatively orconcurrently, individual residues of the human Seg1, Seg2, and/or Seg3sequences can be substituted with corresponding amino acids fromMurineseg1, Murineseg2, and/or Murineseg3 sequences or amino acids withsimilar side chain structure and/or properties to those found in thehuman or murine seg1, seg2, and/or seg3 sequences. For example, thefollowing substitutions can be made:

-   -   1. human p40 with a murine seg1 sequence (residues 97-98 of        murine sequence shown in Table 5, murine sequence insertion)    -   2. human p40 with a murine seg2 sequence (residues 135-142)    -   3. human p40 with a murine seg3 sequence (residues 247-274)    -   4. human p40 with a murine seg1 and seg2 sequence    -   5. human p40 with a murine seg1 and seg3 sequence    -   6. human p40 with a murine seg2 and seg3 sequence    -   7. human p40 with a murine seg1 and seg2 and seg3 sequence    -   8. murine p40 with a human seg1 sequence (corresponding to amino        acids 97-101 of SEQ ID NO:1/sequence in Table 5), human sequence        insertion)    -   9. murine p40 with a human seg2 sequence (residues 138-145 of        SEQ ID NO:1/sequence in Table 5)    -   10. murine p40 with a human seg3 sequence (residues 250-269 of        SEQ ID NO: 1/sequence in Table 5)    -   11. murine p40 with a human seg1 and seg2 sequence    -   12. murine p40 with a human seg1 and seg3 sequence    -   13. murine p40 with a human seg2 and seg3 sequence    -   14. murine p40 with a human seg1 and seg2 and seg3 sequence

The human proteins with murine seg substitutions and the murine proteinswith human seg substitutions can be used for validation and mAbgeneration. For example, proteins #1-7 from above can act as positivevalidation for Abs binding to murine IL-23. Proteins #8-14 can act asnegative validation for such Abs. To validate human IL-23 binding Abs,the use of these two sets of mutants can be reversed.

The described proteins can be utilized to screen and/or select IL-23p40specific binding agents. For example, the described proteins can be usedas immunogens for hybridoma generation. Alternatively, native p40proteins can be used as immunogens and the described proteins can beused for selection of IL-23p40 specific binding agents. Phage displaylibraries can be panned or screened for binding to the describedproteins. Alternatively, the described proteins can be used ascompetitors in a native p40 binding selection process to identifyIL-23p40 specific binding agents. In both cases the described proteinscould be used to generate Abs either as antigens in phage display, forexample, or as screening reagent for mAbs from hybridomas.

In a particular embodiment, to generate anti-human p40 (human IL-23specific mAbs), seg1 in domain 2 can be targeted by phage display. Thereare several schemes that can be adopted. In one scheme, human p40 can beused to select the phage library, and use protein #1 from above (humanp40 with murine Seg1) to screen the Abs, because the Abs reactive towardthis epitope would not bind this chimeric protein and would only bindthe wild type human p40. Abs reactive against both WT and protein #1would not be of interest as they would not be specific for the epitoperegion on p40.

Protein #8 (murine p40 containing human Seg1) may also be used forscreening, because only Abs binding to Seg1 containing epitope wouldpotentially recognize this chimeric molecule assuming the chimericprotein presents an epitope containing Seg1 similar to the human p40.This is generally believed to be the case, since the structural anddifferences are rather small between human and mouse Abs.

In a second embodiment, protein #1 can be used as a competitor in thephage panning to remove all Abs binding human p40 except for thosetargeting the Seg1 containing epitopes. During screening, Abs that bindWT p40 and not protein #1 are the ones of interest and those that bindboth would target other epitopes and not be specific for the epitopesidentified herein (Segs. 1-3). Similar schemes can be applied tohybridoma generation and screening.

The present invention describes IL-23 specific inhibition throughcertain epitopes on the p40 protein. This provides a therapeuticadvantage in that targeting IL-23 will provide significant benefit forimmune-mediated disease. An agent that accomplishes IL-23 inhibition(binding) (i.e., prevent the bioactivity of IL-23) without bindingand/or inhibiting IL-12 would suppress pathogenic biological processesand leave important IL-12 mediated pathogen immunity responses intact.In other words, primary immune responses that are dependent on IL-12would not be affected. Whereas chronic immune responses, such asimmune-mediated disease, that are dependent upon IL-23 would beinhibited. This could provide a therapeutic agent that is applicable toa broader patient population, particularly those patients withimmune-mediated disease that might be at a higher risk for infection.

In addition, the biological actions of IL-12 have been associated withcontrolling tumor growth and eliminating tumor cells. Therefore, IL-23specific inhibition could be a more appropriate alternative for patientswith immune-mediated disease that might be at a higher risk for certaintypes of tumors.

The present invention provides a potency advantage as IL-12Rβ1 isthought to have a high “affinity” binding to IL-12/23p40. Therefore,inhibition of IL-23 p40 and IL-12RP 1 interaction could offer morepotent therapy than agents targeted to IL-23p 19/IL-23R interaction.

Additionally, the present invention provides a cross-reactivityadvantage because, if p40 can associate with alternative proteinsubunits other than p35 or p19, an agent that binds to the described p40epitopes could also bind and neutralize novel p40-based cytokines.

Example 5 Mutant IL-23 Proteins

To confirm the accuracy of the epitope region on the human IL-23p40,referred to above as human seg2 (amino acids 138-145: ₁₃₈RGSSDPQG₁₄₅),mutant mouse and human IL-23p40 proteins were prepared. In the humanIL-23p40, the human seg2 was replaced by the murine seg2 (amino acids135-142: ₁₃₅SSSPDSRA₁₄₂) and, in the murine IL-23p40, the murine seg2was replaced by the human seg2. The mutant and wild-type human andmurine proteins were then tested for binding to the anti-human IL-23 andanti-murine IL-23 antibodies.

Materials

IL-23p40 mutants were transiently-expressed from HEK293E cells. Thedifferent mutants used in this study are listed below:

mIL-23 with hup40Seg2 swap mutant (labeled 3009):

-   -   Murine IL-23 p40 [(mu) ₁₃₅SSSPDSRA₁₄₂→(hu)₁₃₈RGSSDPQG₁₄₅]        hIL-23 with mup40seg2 swap mutant (labeled 3209):    -   Human IL-23 p40 [(hu) ₁₃₈RGSSDPQG₁₄₅→(mu)₁₃₅SSSPDSRA₁₄₂]        murine IL-23 (wild-type)        human IL-23 (wild-type)        The samples were used for binding assays.        Experimental Procedures

MSD high bind plates (Meso Scale Discovery, Gaithersburg, Md.) werecoated with 5 μl of serially diluted samples, from 100 to 0 μg/ml, ofdifferent protein reagents, including human IL-23, murine IL-23 and twoSeg 2-swapped mutant proteins for 2 hours at room temperature. Onehundred fifty (150) μl of 5% MSD Blocker A buffer was added to each welland incubated for 1 hour at room temperature. Plates were washed threetimes with 0.1 M HEPES buffer, pH 7.4. These protein-charged ELISAmicro-wells were incubated with 25 μl of 2 μg/mL MSD Sulfo-TAG labeledCNTO 1836 (anti murine IL-23p40 antibody) or CNTO 1275 mAb(anti-IL-112/IL-23p40 antibody). After incubation for 2 hours withshaking at room temperature, plates were washed 3 times with 0.1 M HEPESbuffer (pH 7.4). MSD Read Buffer T was diluted with distilled water(4-fold) and dispensed at a volume of 150 μl/well and analyzed with aSECTOR imager 6000.

Results:

Previous segment swapping work indicated that the mutated region,(murine) ₁₃₅SSSPDSRA₁₄₂→(human)₁₃₈RGSSDPQG₁₄₅ located at Domain II ofIL-23p40 is the binding epitope on the mouse IL-23p40 protein for CNTO1836. To evaluate the impact of seg2 on IL-23 binding to CNTO 1836,ELISA binding assays were carried out. FIG. 7 shows the binding resultsof these mutant proteins for CNTO 1836. CNTO 1836 did not bind to themurine p40 with the human region corresponding to the seg2 CNTO 1836epitope and the wild-type human IL-23. In contrast, CNTO 1836 did bindto wild-type murine IL-23 and the human p40 with the CNTO 1836 seg2epitope transferred from mouse IL-23 protein.

Additionally, the segment-swapped and wild-type human and murine IL-23proteins were also analyzed for their binding affinity to CNTO 1275.FIG. 8 shows that CNTO 1275 does not bind to murine wild-type IL-23 andthe murine IL-23 swapped mutant (murine p40 with the human regioncorresponding to the CNTO 1836 epitope). In contrast, CNTO 1275 doesbind to human wild-type IL-23 and the human IL-23 swapped mutant (humanp40 with the murine CNTO 1836 epitope inserted in place of the IL-23p40specific epitope). These results confirm that CNTO 1275, which bindsIL-12p40 and IL-23p40 (i.e., it is not selective for IL-23p40), binds tothe first domain of the p40 subunit. This finding is consistent withhuman seg2 being the IL-23p40-specific epitope for an antibody that onlybinds and neutralized IL-23p40 and does not bind and neutralizeIL-12p40.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

TABLE 1 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day −1 (prior to Th1 differentiation). Group IncidenceMortality Day of onset Highest acute cs^(a) Cumul cs^(b) Cs/day No. ofrelapses Relapse severity P-2001-060 Rat IgG 13/13 4/13 30.5 ± 3.2 3.6 ±0.3 71.4 ± 14.1 1.2 ± 0.2 1.3 ± 0.2 3.6 ± 0.2 Anti-p35 11/13 8/13 29.6 ±3.4 3.5 ± 0.5 45.5 ± 11.5 0.8 ± 0.2 1.2 ± 0.1 4.0 ± 0.3 Anti-p40  1/130/13 40.0 0.1 1.2 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 P-2001-079 Notreatment 6/7 0/7  24.7 ± 2.7 3.2 ± 0.6 110.4 ± 20.4  1.7 ± 0.3 1.0 ±0.4 3.8 ± 0.1 Rat IgG 9/9 2/9  29.1 ± 2.9 3.8 ± 0.2 90.6 ± 10.1 1.5 ±0.1 0.3 ± 0.2 4.7 ± 0.3 Anti-p35 10/10 1/10 30.0 ± 2.6 3.9 ± 0.2 94.9 ±17.8 1.4 + 0.2 0.7 ± 0.3 3.9 ± 0.2 Anti-p40  1/10 0/10 61.0 0.3 1.6 ±1.1 0.0 ± 0.0 0.0 + 0.0 0.0 + 0.0 ^(a)clinical score (cs) ^(b)cumulativecs Mice were treated as described and clinical scores were analyzed fromday 0 through 70 days post EAE induction. Data is shown as the mean pergroup ± s.e.m.

TABLE 2 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day 10 (after Th1 differentiation). Highest Relapse GroupIncidence Mortality Day of onset acute cs^(a) Cumul cs^(b) Cs/day #relapses severity P-2001-037 No treatment 7/8 0/8  30.6 ± 2.7 3.2 ± 0.551.5 ± 14.4 0.8 ± 0.2 0.3 ± 0.2 3.3 ± 0.8 Rat IgG  9/10 0/10 25.9 ± 2.72.7 ± 0.5 74.7 ± 15.8 1.2 ± 0.2 0.6 ± 0.2 3.7 ± 0.4 Anti-p35  9/10 0/1025.8 ± 2.6 2.5 ± 0.4 58.8 ± 15.6 1.0 ± 0.2 0.7 ± 0.3 3.2 ± 0.3 Anti-p406/7 0/7  34.7 ± 6.3 1.6 ± 0.5 14.9 ± 7.5  0.2 ± 0.1 0.3 ± 0.2 1.5 ± 0.5P-2001-053 No treatment 8/9 2/9  15.8 ± 2.2 2.1 ± 0.6 56.4 ± 19.1 0.9 ±0.3 0.6 ± 0.3 3.3 ± 0.5 Rat IgG  9/10 4/10 20.0 ± 2.5 3.8 ± 0.5 70.1 ±17.7 1.3 ± 0.2 0.3 ± 0.2 4.2 ± 0.4 Anti-p35 10/10 1/10 16.5 ± 1.1 3.2 ±0.3 93.8 ± 15.7 1.4 ± 0.2 0.8 ± 0.2 3.2 ± 0.3 Anti-p40 10/10 2/10 13.6 ±1.1 2.7 ± 0.5 23.2 ± 7.9  0.4 ± 0.1 0.4 ± 0.3 2.0 ± 0.4 ^(a)clinicalscore (cs) ^(b)cumulative cs Mice were treated on days 10, 17, and 24and clinical scores were analyzed from day 0 through 70 days post EAEinduction. Data is shown as the mean per group ± s.e.m.

TABLE 3 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day 30 (during established EAE). From first treatmentthrough 80 days post EAE induction Group Pre-Tx^(a) Mortality Cumulcs^(b) Cs/day Highest cs Lowest cs # relapses Relapse severity P-2002-01No treatment 2.7 ± 0.6 1/5 132.9 ± 29.3  3.3 ± 0.3 4.1 ± 0.2 2.4 ± 0.50.6 ± 0.4 3.7 ± 0.0 Anti-p35 2.3 ± 0.7 1/5 135.9 ± 16.5  2.7 ± 0.3 3.8 ±0.4 1.8 ± 0.3 2.0 ± 0.4 3.7 ± 0.3 Anti-p40 2.0 ± 0.2 1/6 75.6 ± 16.1 1.9± 0.3 2.8 ± 0.5 1.0 ± 0.4 0.7 ± 0.3 2.5 ± 1.0 P-2002-093 Rat IgG 1.7 ±0.8 1/5 87.7 ± 16.4 2.1 ± 0.2 3.7 ± 0.4 1.2 ± 0.5 1.5 ± 0.5 3.8 ± 1.0Anti-p35 1.9 ± 0.7 1/5 98.2 ± 9.7  2.2 ± 0.1 3.7 ± 0.4 1.4 ± 0.4 1.5 ±0.3 3.3 ± 0.2 Anti-p40 2.4 ± 0.8 0/5 71.7 ± 21.6 1.5 ± 0.4 2.9 ± 0.6 0.8± 0.5 1.3 ± 0.3 2.7 ± 0.6 ^(a)mean clinical score per group on the firstday of treatment (Tx) ^(b)clinical score (cs)

TABLE 4 EAE clinical score analysis with IL-23 specific neutralizationinitiated on day 10 (after Th1 differentiation). Day of Highest No. ofRelapse Group Incid^(a) Mort^(b) Onset Acute cs Cumul cs^(c) Cs/day^(d)relapses severity Expt 1 Rat IgG 9/9 7/9 18.2 ± 0.6 4.7 ± 0.1 47.5 ±15.6 1.1 ± 0.1 0.1 ± 0.0 5.0 ± 0.0 Anti-IL-23 9/9 3/9 28.0 ± 4.6 3.4 ±0.5 46.1 ± 14.9 0.8 ± 0.2 0.4 ± 0.2 2.8 ± 0.4 (20 mg/kg) Anti-IL-23 7/91/9 29.9 ± 4.6 2.8 ± 0.6 57.3 ± 16.9 0.9 ± 0.2 0.3 ± 0.2 3.7 ± 0.2 (50mg/kg) Expt 2 Anti-IL-12   10/10^(e)  4/10 24.2 ± 2.0 3.9 ± 0.4 99.9 ±18.9 1.6 ± 0.3 0.7 ± 0.3 3.4 ± 0.6 Anti-IL-23 9/9 1/9 35.1 ± 3.1 2.8 ±0.5 60.3 ± 16.9 0.9 ± 0.2 0.0 0.0 (20 mg/kg) Anti-IL-23 6/9 1/9 30.7 ±3.3 1.7 ± 0.4 38.1 ± 11.2 0.6 ± 0.2 0.6 ± 0.3 1.9 ± 0.4 (50 mg/kg) Micewere given 3 once weekly doses of Rat IgG or anti-IL-23 starting on day10 post EAE immunization. Clinical scores were analyzed as described inthe Materials and Methods for 70 days post EAE induction. Data is shownas the mean per group ± s.e.m. ^(a)Incidence, ^(b)Mortality,^(c)Cumulative clinical score, ^(d)Clinical score per day.

1. A method of selecting an anti-IL-23 antibody, comprising panning anantibody library with a variant murine p40 subunit of IL-23 andrecovering the antibody binding to the variant murine p40subunit ofIL-23, wherein the variant murine p40 subunit of IL-23 comprises atleast one replacement from the group consisting of: (i) replacingresidues 97 and 98 of the amino acid sequence of SEQ ID NO:2 withresidues 97-101 of the amino acid sequence of SEQ ID NO:1, (ii)replacing residues 135-142 of the amino acid sequence of SEQ ID NO:2with residues 138-145 of the amino acid sequence of SEQ ID NO:1, and(iii) replacing residues 247-274 of the amino acid sequence of SEQ IDNO:2 with residues 250-269 of the amino acid sequence of SEQ ID NO:1. 2.The method of claim 1, wherein the variant murine P40 subunit of IL-23comprises a replacement of residues 135-142 of the amino acid sequenceof SEQ ID NO:2 with residues 138-145 of the amino acid sequence of SEQID NO:1.
 3. The method of claim 1, wherein the variant murine P40subunit of IL-23 comprises a replacement of residues 97 and 98 of theamino acid sequence of SEQ ID NO:2 with residues 97-101 of the aminoacid sequence of SEQ ID NO:1.
 4. The method of claim 3, wherein thevariant murine P40 subunit of IL-23 comprises a replacement of residues247-274 of the amino acid sequence of SEQ ID NO:2 with residues 250-269of the amino acid sequence of SEQ ID NO:1.
 5. The method of claim 1,wherein the variant murine P40 subunit of IL-23 comprises: (i) areplacement of residues 97 and 98 of the amino acid sequence of SEQ IDNO:2 with residues 97-101 of the amino acid sequence of SEQ ID NO:1,(ii) a replacement of residues 135-142 of the amino acid sequence of SEQID NO:2 with residues 138-145 of the amino acid sequence of SEQ ID NO:1,and (iii) a replacement of residues 247-274 of the amino acid sequenceof SEQ ID NO:2 with residues 250-269 of the amino acid sequence of SEQID NO:1.
 6. A method of generating an anti-IL-23 antibody, comprisingimmunizing an animal producing antibodies with a variant murine p40subunit of IL-23 and recovering an antibody binding to the variantmurine p40 subunit of IL-23, wherein the variant murine p40 subunit ofIL-23 comprises at least one replacement from the group consisting of(i) replacing residues 97 and 98 of the amino acid sequence of SEQ IDNO:2 with residues 97-101 of the amino acid sequence of SEQ ID NO:1,(ii) replacing residues 135-142 of the amino acid sequence of SEQ IDNO:2 with residues 138-145 of the amino acid sequence of SEQ ID NO:1,and (iii) replacing residues 247-274 of the amino acid sequence of SEQID NO:2 with residues 250-269 of the amino acid sequence of SEQ ID NO:1.7. The method of claim 6, wherein the variant murine P40 subunit ofIL-23 comprises a replacement of residues 135-142 of the amino acidsequence of SEQ ID NO:2 with residues 138-145 of the amino acid sequenceof SEQ ID NO:1.
 8. The method of claim 6, wherein the variant murine P40subunit of IL-23 comprises a replacement of residues 97 and 98 of theamino acid sequence of SEQ ID NO:2 with residues 97-101 of the aminoacid sequence of SEQ ID NO:1.
 9. The method of claim 6, wherein thevariant murine P40 subunit of IL-23 comprises a replacement of residues247-274 of the amino acid sequence of SEQ ID NO:2 with residues 250-269of the amino acid sequence of SEQ ID NO:1.
 10. The method of claim 6,wherein the variant murine p40 subunit of IL-23 comprises: (i) areplacement of residues 97 and 98 of the amino acid sequence of SEQ IDNO:2 with residues 97-101 of the amino acid sequence of SEQ ID NO:1,(ii) a replacement of residues 135-142 of the amino acid sequence of SEQID NO:2 with residues 138-145 of the amino acid sequence of SEQ ID NO:1,and (iii) a replacement of residues 247-274 of the amino acid sequenceof SEQ ID NO:2 with residues 250-269 of the amino acid sequence of SEQID NO:1.